U.S. patent number 6,037,176 [Application Number 09/344,520] was granted by the patent office on 2000-03-14 for antisense inhibition of integrin beta 3 expression.
This patent grant is currently assigned to Isis Pharmaceuticals Inc.. Invention is credited to C. Frank Bennett, Lex M. Cowsert, Brett P. Monia.
United States Patent |
6,037,176 |
Bennett , et al. |
March 14, 2000 |
**Please see images for:
( Certificate of Correction ) ** |
Antisense inhibition of integrin beta 3 expression
Abstract
Antisense compounds, compositions and methods are provided for
modulating the expression of integrin beta 3. The compositions
comprise antisense compounds, particularly antisense
oligonucleotides, targeted to nucleic acids encoding integrin beta
3. Methods of using these compounds for modulation of integrin beta
3 expression and for treatment of diseases associated with
expression of integrin beta 3 are provided.
Inventors: |
Bennett; C. Frank (Carlsbad,
CA), Monia; Brett P. (La Costa, CA), Cowsert; Lex M.
(Carlsbad, CA) |
Assignee: |
Isis Pharmaceuticals Inc.
(Carlsbad, CA)
|
Family
ID: |
23350871 |
Appl.
No.: |
09/344,520 |
Filed: |
June 25, 1999 |
Current U.S.
Class: |
435/375; 435/325;
435/366; 435/455; 435/6.16; 435/91.1; 536/23.1; 536/24.31;
536/24.33; 536/24.5 |
Current CPC
Class: |
C12N
15/1138 (20130101); A61K 38/00 (20130101); A61K
48/00 (20130101); C12N 2310/315 (20130101); C12N
2310/321 (20130101); C12N 2310/3341 (20130101); C12N
2310/341 (20130101); C12N 2310/346 (20130101); C12N
2310/321 (20130101); C12N 2310/3525 (20130101); Y02P
20/582 (20151101) |
Current International
Class: |
C12N
15/11 (20060101); A61K 38/00 (20060101); A61K
48/00 (20060101); C12Q 001/68 (); C12N 015/00 ();
C07H 021/04 () |
Field of
Search: |
;435/6,91.1,325,366,375,455 ;536/23.1,24.31,24.33,24.5 ;514/44 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Dallahrida et al. FASEB Journal. 12(8):pA1346, Apr. 24, 1998. .
Dallahrida et al. Blood 90(10), Suppl. 1 (Part 1 of 2), p286a,
1261, Nov. 15, 1997. .
James, W. Antiviral Chemistry Chemotherapy 2:191-214 (1991). .
Kumer et al. J. Biol. Chem. 272(26):16390-16397, 1997. .
Milner et al. Nature Biotechnology 15:537-541, Jun. 1997. .
Branch TIBS 23:45-50 1998. .
Crooke S.T. ed. "Antisense Research and Application", Ch1, pp.
1-50, Springer-Verlag, New York 1998. .
Akiyama, Integrins in cell adhesion and signaling, Hum. Cell, 1996,
9:181-186. .
Cheresh, Structure, function and biological properties of integrin
alpha v beta 3 on human melanoma cells, Cancer Metastasis Rev.,
1991, 10:3-10. .
Clemetson et al., Integrins and cardiovascular disease, Cell. Mol.
Life Sci., 1998, 54:502-513. .
Elangbam et al., Cell adhesion molecules--undate, Vet. Pathol.,
1997, 34:61-73. .
Horton, The alpha v beta 3 integrin "vitronectin receptor", Int. J.
Biochem. Cell. Biol., 1997, 29:721-725. .
Kato, The biologic and clinical spectrum of Glanzmann's
thrombasthenia: implications of integrin alpha IIb beta 3 for its
pathogenesis, Crit. Rev. Oncol. Hematol., 1997, 26:1-23. .
LaFlamme et al., Integrin cytoplasmic domains as connectors to the
cell's signal tranduction apparatus, Matrix Biol., 1997,
16:153-163. .
Rodan et al., Integrin function in osteoclasts, J. Endocrinol.,
1997, 154 Suppl:S47-56. .
Ruoslahti, Integrins as signaling molecules and targets for tumor
therapy, Kidney Int., 1997, 51:1413-1417. .
Shattil, Regulation of platelet anchorage and signaling by integrin
alpha IIb beta 3, Thromb. Haemost., 1993, 70:224-228. .
Trikha et al., Human prostate carcinoma cells express functional
alphaIIb (beta) 3 integrin, Cancer Res., 1996,
56:5071-5078..
|
Primary Examiner: LeGuyader; John L.
Attorney, Agent or Firm: Law Offices of Jane Massey
Licata
Claims
What is claimed is:
1. An antisense compound up to 30 nucleobases in length comprising
at least an 8-nucleobase portion of SEQ ID NO: 8, 10, 11, 12, 15,
17, 18, 21, 22, 23, 24, 25, 26, 27, 30, 31, 35, 36, 38, 42, 43, 44,
46, 47, 9, 13, 20, 28, 29, 33 or 41 which inhibits the expression
of human integrin beta 3, SEQ ID NO: 1.
2. The antisense compound of claim 1 which is an antisense
oligonucleotide.
3. The antisense compound of claim 2 wherein the antisense
oligonucleotide comprises at least one modified internucleoside
linkage.
4. The antisense compound of claim 3 wherein the modified
internucleoside linkage is a phosphorothioate linkage.
5. The antisense compound of claim 2 wherein the antisense
oligonucleotide comprises at least one modified sugar moiety.
6. The antisense compound of claim 5 wherein the modified sugar
moiety is a 2'-O-methoxyethyl sugar moiety.
7. The antisense compound of claim 2 wherein the antisense
oligonucleotide comprises at least one modified nucleobase.
8. The antisense compound of claim 7 wherein the modified
nucleobase is a 5-methylcytosine.
9. The antisense compound of claim 2 wherein the antisense
oligonucleotide is a chimeric oligonucleotide.
10. A method of inhibiting the expression of human integrin beta 3
in human cells or tissues comprising contacting said cells or
tissues in vitro with the antisense compound of claim 1 so that
expression of human integrin beta 3, SEQ ID NO: 1, is inhibited.
Description
FIELD OF THE INVENTION
The present invention provides compositions and methods for
modulating the expression of integrin beta 3. In particular, this
invention relates to antisense compounds, particularly
oligonucleotides, specifically hybridizable with nucleic acids
encoding human integrin beta 3. Such oligonucleotides have been
shown to modulate the expression of integrin beta 3.
BACKGROUND OF THE INVENTION
Cell adhesive contacts are critical for the development and
maintenance of multicellular organisms. These contacts are mediated
by cell adhesion molecules (CAMs), a versatile class of compounds
expressed on the cell surface. Cells adhere to one another and to
extracellular substrates through the concerted action of a variety
of CAMs, which act as both receptors and ligands on opposing cells.
There are four subclasses of CAMs; selectins, cadherins,
immunoglobulins and integrins. Selectins influence the localization
of circulating leukocytes during inflammation, while cadherins and
immunoglobulins establish and maintain cell-to-cell association and
recognition mechanisms (Elangbam et al., Vet. Pathol., 1997, 34,
61-73). The fourth class of CAMs, known as integrins, play an
important role in cell migration, cell anchorage to substrates and
cytoadhesion signaling pathways (Akiyama, Hum. Cell, 1996, 9,
181-186).
Integrins are heterodimeric cation-dependent membrane glycoproteins
composed of an alpha and beta subunit. To date, 8 beta and 15 alpha
subunits have been identified and these subunits have been shown to
combine to form over 20 different .alpha..beta. heterodimers.
Integrins have been found in all tissues examined and consist of a
large extracellular domain, a transmembrane domain and a smaller
cytoplasmic domain. It is the extracellular domain of the integrin
that acts as a receptor for various matrix proteins, while the
cytoplasmic domain has been shown to interact with actin filaments
of the cytoskeleton, thereby mediating signaling cascades (LaFlamme
et al., Matrix Biol., 1997, 16, 153-163).
Integrin beta 3 (also known as human endothelial glycoprotein,
GP3A, GPIIIa, ITGB3, CD61 and platelet glycoprotein 3a) is the
common beta subunit partner of the members of the .beta.3 subfamily
of integrins. This subfamily consists of two members, the
vitronectin receptor, and the fibrinogen receptor and cells
expressing this class of integrin receptor can adhere to various
matrix proteins and participate in cytoadhesion driven cellular
responses.
Integrin beta 3, in conjunction with integrin alpha v, forms the
vitronectin receptor (.alpha.V.beta.3). This heterodimeric receptor
is localized to platelets, endothelial cells, monocytes,
macrophages and osteoclasts with the highest expression found in
the osteoclasts (Rodan and Rodan, J. Endocrinol., 1997, 154 Suppl,
S47-56). The vitronectin receptor functions to mediate the adhesion
of cells to vitronectin, and a variety of extracellular matrix
proteins. It is through a specific tripeptide sequence referred to
as the RGD sequence, so named because of its amino acid composition
(arginine-glycine-aspartic acid), that receptor-protein binding
occurs. One category of inhibitors that targets the vitronectin
receptor are peptidomimetics, designed to block the interactions
between the receptor and RGD-containing proteins (Akiyama, Hum.
Cell, 1996, 9, 181-186; Horton, Int. J. Biochem. Cell. Biol., 1997,
29, 721-725).
The activation of the vitronectin receptor has been shown to
promote cellular migration and to provide signals in the regulation
of cell proliferation and differentiation and to potentiate the
effects of insulin (Ruoslahti, Kidney Int., 1997, 51, 1413-1417).
Upregulation of the vitronectin receptor is associated with
pathological conditions such as vascular restinosis (Clemetson and
Clemetson, Cell. Mol. Life Sci., 1998, 54, 502-513), excessive bone
resorption (Rodan and Rodan, J. Endocrinol., 1997, 154 Suppl,
S47-56), and the process of angiogenesis during malignant melanomas
(Cheresh, Cancer Metastasis Rev., 1991, 10, 3-10).
Integrin beta 3, in conjunction with integrin alpha IIb, also forms
the fibrinogen receptor (.alpha.IIb/.beta.3) which mediates
platelet aggregation. This receptor is basally inactive but can be
activated by several agonists causing it to bind fibrinogen which
then forms cross-bridges to fibrinogen receptors on adjacent cells.
This receptor has also been shown to bind other proteins including
fibronectin, von Willebrand factor (vWf) and vitronectin (Shattil,
Thromb. Haemost., 1993, 70, 224-228).
Mutations in the gene of either subunit of the fibrinogen receptor,
resulting in receptor deletion or misfunction, are the primary
cause of the bleeding disorder known as Glanzman's thrombasthenia
(GT) (Kato, Crit. Rev. Oncol. Hematol., 1997, 26, 1-23). This
disorder is transmitted as an autosomal recessive trait and several
mutations, including gene deletions, point mutations, and
rearrangements have been identified (Kato, Crit. Rev. Oncol.
Hematol., 1997, 26, 1-23).
Currently, therapeutic agents which affect the function of
receptors containing the integrin beta 3 subunit have been designed
to interfere with the binding properties of the receptor. As such,
several inhibitors have been reported in the art and these include
synthetic compounds and their derivatives, antibodies, and
peptidomimetics, all of which act as antagonists to receptor ligand
binding.
Abciximab, ReoPro.RTM. (Eli Lilly and Co.) is the Fab fragment of
the chimeric human-murine monoclonal antibody 7E3. Abciximab binds
to the glycoprotein (GP) IIb/IIIa (.alpha.IIb.beta.3) receptor of
human platelets and inhibits platelet aggregation. The mechanism of
action is thought to involve steric hindrance and/or conformational
effects to block access of large molecules to the receptor rather
than direct interaction with the RGD (arginine-glycine-aspartic
acid) binding site of GPIIb/IIIa. Abciximab also binds with similar
affinity to the vitronectin (.alpha.v.beta.3) receptor found on
platelets and vessel wall endothelial and smooth muscle cells.
Abciximab is indicated as an adjunct to percutaneous coronary
intervention for the prevention of cardiac ischemic complications.
However, a potentially dangerous human antibody response to the
chimeric antibody occurred in approximately 6% of patients
(Clemetson and Clemetson, Cell. Mol. Life Sci., 1998, 54,
502-513).
Monoclonal antibodies to both integrin beta 3-containing receptors
have been reported in the literature. Recently it was demonstrated
that prostate carcinoma cells express the .alpha.IIb/.beta.3
fibrinogen receptor and that antibodies to this receptor were
capable of inhibiting the invasive properties of the carcinomal
cells (Trikha et al., Cancer Res., 1996, 56, 5071-5078).
Monoclonal antibodies produced by three different hybridoma cell
lines that target the .alpha.v.beta.3 (vitronectin) receptor and
inhibit the binding of fibronectin or vitronectin to osteoclasts
are disclosed in U.S. Pat. Nos. 5,578,704, 5,652,109 and 5,652,110.
These antibodies are used to detect the .alpha.V.beta.3 integrin
and to treat disease conditions characterized by excessive bone
resorption (Kim et al., 1997,; Kim et al., 1997,; Kim et al.,
1996).
To date, no therapeutic agents that effectively inhibit the
expression of integrin beta 3 have been identified. Consequently,
there remains a long felt need for additional agents capable of
effectively inhibiting integrin beta 3 function by reducing the
levels of expression.
Antisense technology is emerging as an effective means for reducing
the expression of specific gene products and may therefore prove to
be uniquely useful in a number of therapeutic, diagnostic, and
research applications for the modulation of integrin beta 3
expression.
SUMMARY OF THE INVENTION
The present invention is directed to antisense compounds,
particularly oligonucleotides, which are targeted to a nucleic acid
encoding integrin beta 3, and which modulate the expression of
integrin beta 3. Pharmaceutical and other compositions comprising
the antisense compounds of the invention are also provided. Further
provided are methods of modulating the expression of integrin beta
3 in cells or tissues comprising contacting said cells or tissues
with one or more of the antisense compounds or compositions of the
invention. Further provided are methods of treating an animal,
particularly a human, suspected of having or being prone to a
disease or condition associated with expression of integrin beta 3
by administering a therapeutically or prophylactically effective
amount of one or more of the antisense compounds or compositions of
the invention.
DETAILED DESCRIPTION OF THE INVENTION
The present invention employs oligomeric antisense compounds,
particularly oligonucleotides, for use in modulating the function
of nucleic acid molecules encoding integrin beta 3, ultimately
modulating the amount of integrin beta 3 produced. This is
accomplished by providing antisense compounds which specifically
hybridize with one or more nucleic acids encoding integrin beta 3.
As used herein, the terms "target nucleic acid" and "nucleic acid
encoding integrin beta 3" encompass DNA encoding integrin beta 3,
RNA (including pre-mRNA and mRNA) transcribed from such DNA, and
also cDNA derived from such RNA. The specific hybridization of an
oligomeric compound with its target nucleic acid interferes with
the normal function of the nucleic acid. This modulation of
function of a target nucleic acid by compounds which specifically
hybridize to it is generally referred to as "antisense". The
functions of DNA to be interfered with include replication and
transcription. The functions of RNA to be interfered with include
all vital functions such as, for example, translocation of the RNA
to the site of protein translation, translation of protein from the
RNA, splicing of the RNA to yield one or more mRNA species, and
catalytic activity which may be engaged in or facilitated by the
RNA. The overall effect of such interference with target nucleic
acid function is modulation of the expression of integrin beta 3.
In the context of the present invention, "modulation" means either
an increase (stimulation) or a decrease (inhibition) in the
expression of a gene. In the context of the present invention,
inhibition is the preferred form of modulation of gene expression
and mRNA is a preferred target.
It is preferred to target specific nucleic acids for antisense.
"Targeting" an antisense compound to a particular nucleic acid, in
the context of this invention, is a multistep process. The process
usually begins with the identification of a nucleic acid sequence
whose function is to be modulated. This may be, for example, a
cellular gene (or mRNA transcribed from the gene) whose expression
is associated with a particular disorder or disease state, or a
nucleic acid molecule from an infectious agent. In the present
invention, the target is a nucleic acid molecule encoding integrin
beta 3. The targeting process also includes determination of a site
or sites within this gene for the antisense interaction to occur
such that the desired effect, e.g., detection or modulation of
expression of the protein, will result. Within the context of the
present invention, a preferred intragenic site is the region
encompassing the translation initiation or termination codon of the
open reading frame (ORF) of the gene. Since, as is known in the
art, the translation initiation codon is typically 5'-AUG (in
transcribed mRNA molecules; 5'-ATG in the corresponding DNA
molecule), the translation initiation codon is also referred to as
the "AUG codon," the "start codon" or the "AUG start codon". A
minority of genes have a translation initiation codon having the
RNA sequence 5'-GUG, 5'-UUG or 5'-CUG, and 5'-AUA, 5'-ACG and
5'-CUG have been shown to function in vivo. Thus, the terms
"translation initiation codon" and "start codon" can encompass many
codon sequences, even though the initiator amino acid in each
instance is typically methionine (in eukaryotes) or
formylmethionine (in prokaryotes). It is also known in the art that
eukaryotic and prokaryotic genes may have two or more alternative
start codons, any one of which may be preferentially utilized for
translation initiation in a particular cell type or tissue, or
under a particular set of conditions. In the context of the
invention, "start codon" and "translation initiation codon" refer
to the codon or codons that are used in vivo to initiate
translation of an mRNA molecule transcribed from a gene encoding
integrin beta 3, regardless of the sequence(s) of such codons.
It is also known in the art that a translation termination codon
(or "stop codon") of a gene may have one of three sequences, i.e.,
5'-UAA, 5'-UAG and 5'-UGA (the corresponding DNA sequences are
5'-TAA, 5'-TAG and 5'-TGA, respectively). The terms "start codon
region" and "translation initiation codon region" refer to a
portion of such an mRNA or gene that encompasses from about 25 to
about 50 contiguous nucleotides in either direction (i.e., 5' or
3') from a translation initiation codon. Similarly, the terms "stop
codon region" and "translation termination codon region" refer to a
portion of such an mRNA or gene that encompasses from about 25 to
about 50 contiguous nucleotides in either direction (i.e., 5' or
3') from a translation termination codon.
The open reading frame (ORF) or "coding region," which is known in
the art to refer to the region between the translation initiation
codon and the translation termination codon, is also a region which
may be targeted effectively. Other target regions include the 5'
untranslated region (5'UTR), known in the art to refer to the
portion of an mRNA in the 5' direction from the translation
initiation codon, and thus including nucleotides between the 5' cap
site and the translation initiation codon of an mRNA or
corresponding nucleotides on the gene, and the 3' untranslated
region (3'UTR), known in the art to refer to the portion of an mRNA
in the 3' direction from the translation termination codon, and
thus including nucleotides between the translation termination
codon and 3' end of an mRNA or corresponding nucleotides on the
gene. The 5' cap of an mRNA comprises an N7-methylated guanosine
residue joined to the 5'-most residue of the mRNA via a 5'-5'
triphosphate linkage. The 5' cap region of an mRNA is considered to
include the 5' cap structure itself as well as the first 50
nucleotides adjacent to the cap. The 5' cap region may also be a
preferred target region.
Although some eukaryotic mRNA transcripts are directly translated,
many contain one or more regions, known as "introns," which are
excised from a transcript before it is translated. The remaining
(and therefore translated) regions are known as "exons" and are
spliced together to form a continuous mRNA sequence. mRNA splice
sites, i.e., intron-exon junctions, may also be preferred target
regions, and are particularly useful in situations where aberrant
splicing is implicated in disease, or where an overproduction of a
particular mRNA splice product is implicated in disease. Aberrant
fusion junctions due to rearrangements or deletions are also
preferred targets. It has also been found that introns can also be
effective, and therefore preferred, target regions for antisense
compounds targeted, for example, to DNA or pre-mRNA.
Once one or more target sites have been identified,
oligonucleotides are chosen which are sufficiently complementary to
the target, i.e., hybridize sufficiently well and with sufficient
specificity, to give the desired effect.
In the context of this invention, "hybridization" means hydrogen
bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen
hydrogen bonding, between complementary nucleoside or nucleotide
bases. For example, adenine and thymine are complementary
nucleobases which pair through the formation of hydrogen bonds.
"Complementary," as used herein, refers to the capacity for precise
pairing between two nucleotides. For example, if a nucleotide at a
certain position of an oligonucleotide is capable of hydrogen
bonding with a nucleotide at the same position of a DNA or RNA
molecule, then the oligonucleotide and the DNA or RNA are
considered to be complementary to each other at that position. The
oligonucleotide and the DNA or RNA are complementary to each other
when a sufficient number of corresponding positions in each
molecule are occupied by nucleotides which can hydrogen bond with
each other. Thus, "specifically hybridizable" and "complementary"
are terms which are used to indicate a sufficient degree of
complementarity or precise pairing such that stable and specific
binding occurs between the oligonucleotide and the DNA or RNA
target. It is understood in the art that the sequence of an
antisense compound need not be 100% complementary to that of its
target nucleic acid to be specifically hybridizable. An antisense
compound is specifically hybridizable when binding of the compound
to the target DNA or RNA molecule interferes with the normal
function of the target DNA or RNA to cause a loss of utility, and
there is a sufficient degree of complementarity to avoid
non-specific binding of the antisense compound to non-target
sequences under conditions in which specific binding is desired,
i.e., under physiological conditions in the case of in vivo assays
or therapeutic treatment, and in the case of in vitro assays, under
conditions in which the assays are performed.
Antisense compounds are commonly used as research reagents and
diagnostics. For example, antisense oligonucleotides, which are
able to inhibit gene expression with exquisite specificity, are
often used by those of ordinary skill to elucidate the function of
particular genes. Antisense compounds are also used, for example,
to distinguish between functions of various members of a biological
pathway. Antisense modulation has, therefore, been harnessed for
research use.
The specificity and sensitivity of antisense is also harnessed by
those of skill in the art for therapeutic uses. Antisense
oligonucleotides have been employed as therapeutic moieties in the
treatment of disease states in animals and man. Antisense
oligonucleotides have been safely and effectively administered to
humans and numerous clinical trials are presently underway. It is
thus established that oligonucleotides can be useful therapeutic
modalities that can be configured to be useful in treatment regimes
for treatment of cells, tissues and animals, especially humans. In
the context of this invention, the term "oligonucleotide" refers to
an oligomer or polymer of ribonucleic acid (RNA) or
deoxyribonucleic acid (DNA) or mimetics thereof. This term includes
oligonucleotides composed of naturally-occurring nucleobases,
sugars and covalent internucleoside (backbone) linkages as well as
oligonucleotides having non-naturally-occurring portions which
function similarly. Such modified or substituted oligonucleotides
are often preferred over native forms because of desirable
properties such as, for example, enhanced cellular uptake, enhanced
affinity for nucleic acid target and increased stability in the
presence of nucleases.
While antisense oligonucleotides are a preferred form of antisense
compound, the present invention comprehends other oligomeric
antisense compounds, including but not limited to oligonucleotide
mimetics such as are described below. The antisense compounds in
accordance with this invention preferably comprise from about 8 to
about 30 nucleobases (i.e. from about 8 to about 30 linked
nucleosides). Particularly preferred antisense compounds are
antisense oligonucleotides, even more preferably those comprising
from about 12 to about 25 nucleobases. As is known in the art, a
nucleoside is a base-sugar combination. The base portion of the
nucleoside is normally a heterocyclic base. The two most common
classes of such heterocyclic bases are the purines and the
pyrimidines. Nucleotides are nucleosides that further include a
phosphate group covalently linked to the sugar portion of the
nucleoside. For those nucleosides that include a pentofuranosyl
sugar, the phosphate group can be linked to either the 2', 3' or 5'
hydroxyl moiety of the sugar. In forming oligonucleotides, the
phosphate groups covalently link adjacent nucleosides to one
another to form a linear polymeric compound. In turn the respective
ends of this linear polymeric structure can be further joined to
form a circular structure, however, open linear structures are
generally preferred. Within the oligonucleotide structure, the
phosphate groups are commonly referred to as forming the
internucleoside backbone of the oligonucleotide. The normal linkage
or backbone of RNA and DNA is a 3' to 5' phosphodiester
linkage.
Specific examples of preferred antisense compounds useful in this
invention include oligonucleotides containing modified backbones or
non-natural internucleoside linkages. As defined in this
specification, oligonucleotides having modified backbones include
those that retain a phosphorus atom in the backbone and those that
do not have a phosphorus atom in the backbone. For the purposes of
this specification, and as sometimes referenced in the art,
modified oligonucleotides that do not have a phosphorus atom in
their internucleoside backbone can also be considered to be
oligonucleosides.
Preferred modified oligonucleotide backbones include, for example,
phosphorothioates, chiral phosphorothioates, phosphorodithioates,
phosphotriesters, aminoalkylphosphotriesters, methyl and other
alkyl phosphonates including 3'-alkylene phosphonates and chiral
phosphonates, phosphinates, phosphoramidates including 3'-amino
phosphoramidate and aminoalkylphosphoramidates,
thionophosphoramidates, thionoalkylphosphonates,
thionoalkylphosphotriesters, and boranophosphates having normal
3'-5' linkages, 2'-5' linked analogs of these, and those having
inverted polarity wherein the adjacent pairs of nucleoside units
are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed
salts and free acid forms are also included.
Representative United States patents that teach the preparation of
the above phosphorus-containing linkages include, but are not
limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301;
5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302;
5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233;
5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;
5,563,253; 5,571,799; 5,587,361; and 5,625,050, certain of which
are commonly owned with this application, and each of which is
herein incorporated by reference.
Preferred modified oligonucleotide backbones that do not include a
phosphorus atom therein have backbones that are formed by short
chain alkyl or cycloalkyl internucleoside linkages, mixed
heteroatom and alkyl or cycloalkyl internucleoside linkages, or one
or more short chain heteroatomic or heterocyclic internucleoside
linkages. These include those having morpholino linkages (formed in
part from the sugar portion of a nucleoside); siloxane backbones;
sulfide, sulfoxide and sulfone backbones; formacetyl and
thioformacetyl backbones; methylene formacetyl and thioformacetyl
backbones; alkene containing backbones; sulfamate backbones;
methyleneimino and methylenehydrazino backbones; sulfonate and
sulfonamide backbones; amide backbones; and others having mixed N,
O, S and CH.sub.2 component parts.
Representative United States patents that teach the preparation of
the above oligonucleosides include, but are not limited to, U.S.
Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141;
5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677;
5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240;
5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070;
5,663,312; 5,633,360; 5,677,437; and 5,677,439, certain of which
are commonly owned with this application, and each of which is
herein incorporated by reference.
In other preferred oligonucleotide mimetics, both the sugar and the
internucleoside linkage, i.e., the backbone, of the nucleotide
units are replaced with novel groups. The base units are maintained
for hybridization with an appropriate nucleic acid target compound.
One such oligomeric compound, an oligonucleotide mimetic that has
been shown to have excellent hybridization properties, is referred
to as a peptide nucleic acid (PNA). In PNA compounds, the
sugar-backbone of an oligonucleotide is replaced with an amide
containing backbone, in particular an aminoethylglycine backbone.
The nucleobases are retained and are bound directly or indirectly
to aza nitrogen atoms of the amide portion of the backbone.
Representative United States patents that teach the preparation of
PNA compounds include, but are not limited to, U.S. Pat. Nos.
5,539,082; 5,714,331; and 5,719,262, each of which is herein
incorporated by reference. Further teaching of PNA compounds can be
found in Nielsen et al., Science, 1991, 254, 1497-1500.
Most preferred embodiments of the invention are oligonucleotides
with phosphorothioate backbones and oligonucleosides with
heteroatom backbones, and in particular --CH.sub.2
--NH--O--CH.sub.2 --, --CH.sub.2 --N(CH.sub.3)--O--CH.sub.2 --
[known as a methylene (methylimino) or MMI backbone], --CH.sub.2
--O--N(CH.sub.3)--CH.sub.2 --, --CH.sub.2
--N(CH.sub.3)--N(CH.sub.3)--CH.sub.2 -- and
--O--N(CH.sub.3)--CH.sub.2 --CH.sub.2 -- [wherein the native
phosphodiester backbone is represented as --O--P--O--CH.sub.2 --]
of the above referenced U.S. Pat. No. 5,489,677, and the amide
backbones of the above referenced U.S. Pat. No. 5,602,240. Also
preferred are oligonucleotides having morpholino backbone
structures of the above-referenced U.S. Pat. No. 5,034,506.
Modified oligonucleotides may also contain one or more substituted
sugar moieties. Preferred oligonucleotides comprise one of the
following at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or
N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the
alkyl, alkenyl and alkynyl may be substituted or unsubstituted
C.sub.1 to C.sub.10 alkyl or C.sub.2 to C.sub.10 alkenyl and
alkynyl. Particularly preferred are O[(CH.sub.2).sub.n O].sub.m
CH.sub.3, O(CH.sub.2).sub.n OCH.sub.3, O(CH.sub.2).sub.n NH.sub.2,
O(CH.sub.2).sub.n CH.sub.3, O(CH.sub.2).sub.n ONH.sub.2, and
O(CH.sub.2).sub.n ON[(CH.sub.2).sub.n CH.sub.3)].sub.2, where n and
m are from 1 to about 10. Other preferred oligonucleotides comprise
one of the following at the 2' position: C.sub.1 to C.sub.10 lower
alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or
O-aralkyl, SH, SCH.sub.3, OCN, Cl, Br, CN, CF.sub.3, OCF.sub.3,
SOCH.sub.3, SO.sub.2 CH.sub.3, ONO.sub.2, NO.sub.2, N.sub.3,
NH.sub.2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino,
polyalkylamino, substituted silyl, an RNA cleaving group, a
reporter group, an intercalator, a group for improving the
pharmacokinetic properties of an oligonucleotide, or a group for
improving the pharmacodynamic properties of an oligonucleotide, and
other substituents having similar properties. A preferred
modification includes 2'-methoxyethoxy (2'-O--CH.sub.2 CH.sub.2
OCH.sub.3, also known as 2'-O--(2-methoxyethyl) or 2'-MOE) (Martin
et al., Helv. Chim. Acta, 1995, 78, 486-504) i.e., an alkoxyalkoxy
group. A further preferred modification includes
2'-dimethylaminooxyethoxy, i.e., a O(CH.sub.2).sub.2
ON(CH.sub.3).sub.2 group, also known as 2'-DMAOE, as described in
examples hereinbelow, and 2'-dimethylaminoethoxyethoxy (also known
in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e.,
2'-O--CH.sub.2 --O--CH.sub.2 --N(CH.sub.2).sub.2, also described in
examples hereinbelow.
Other preferred modifications include 2'-methoxy (2'-O--CH.sub.3),
2'-aminopropoxy (2'-OCH.sub.2 CH.sub.2 CH.sub.2 NH.sub.2) and
2'-fluoro (2'-F). Similar modifications may also be made at other
positions on the oligonucleotide, particularly the 3' position of
the sugar on the 3' terminal nucleotide or in 2'-5' linked
oligonucleotides and the 5' position of 5' terminal nucleotide.
Oligonucleotides may also have sugar mimetics such as cyclobutyl
moieties in place of the pentofuranosyl sugar. Representative
United States patents that teach the preparation of such modified
sugar structures include, but are not limited to, U.S. Pat. Nos.
4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137;
5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722;
5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873;
5,670,633; and 5,700,920, certain of which are commonly owned with
the instant application, and each of which is herein incorporated
by reference in its entirety.
Oligonucleotides may also include nucleobase (often referred to in
the art simply as "base") modifications or substitutions. As used
herein, "unmodified" or "natural" nucleobases include the purine
bases adenine (A) and guanine (G), and the pyrimidine bases thymine
(T), cytosine (C) and uracil (U). Modified nucleobases include
other synthetic and natural nucleobases such as 5-methylcytosine
(5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine,
2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and
guanine, 2-propyl and other alkyl derivatives of adenine and
guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,
5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo
uracil, cytosine and thymine, 5-uracil (pseudouracil),
4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and
other 8-substituted adenines and guanines, 5-halo particularly
5-bromo, 5-trifluoromethyl and other 5-substituted uracils and
cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and
8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine
and 3-deazaadenine. Further nucleobases include those disclosed in
U.S. Pat. No. 3,687,808, those disclosed in The Concise
Encyclopedia Of Polymer Science And Engineering, pages 858-859,
Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed
by Englisch et al., Angewandte Chemie, International Edition, 1991,
30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15,
Antisense Research and Applications, pages 289-302, Crooke, S. T.
and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases
are particularly useful for increasing the binding affinity of the
oligomeric compounds of the invention. These include 5-substituted
pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted
purines, including 2-aminopropyladenine, 5-propynyluracil and
5-propynylcytosine. 5-methylcytosine substitutions have been shown
to increase nucleic acid duplex stability by 0.6-1.2.degree. C.
(Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense
Research and Applications, CRC Press, Boca Raton, 1993, pp.
276-278) and are presently preferred base substitutions, even more
particularly when combined with 2'-O-methoxyethyl sugar
modifications.
Representative United States patents that teach the preparation of
certain of the above noted modified nucleobases as well as other
modified nucleobases include, but are not limited to, the above
noted U.S. Pat. No. 3,687,808, as well as U.S. Pat. Nos. 4,845,205;
5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187;
5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469;
5,594,121, 5,596,091; 5,614,617; and 5,681,941, certain of which
are commonly owned with the instant application, and each of which
is herein incorporated by reference, and U.S. Pat. No. 5,750,692,
which is commonly owned with the instant application and also
herein incorporated by reference.
Another modification of the oligonucleotides of the invention
involves chemically linking to the oligonucleotide one or more
moieties or conjugates which enhance the activity, cellular
distribution or cellular uptake of the oligonucleotide. Such
moieties include but are not limited to lipid moieties such as a
cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA,
1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med.
Chem. Let., 1994, 4, 1053-1060), a thioether, e.g.,
hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,
660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3,
2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,
1992, 20, 533-538), an aliphatic chain, e.g., dodecandiol or
undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10,
1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330;
Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,
e.g., di-hexadecyl-rac-glycerol or triethylammonium
1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,
Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids
Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol
chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14,
969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron
Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al.,
Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine
or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J.
Pharmacol. Exp. Ther., 1996, 277, 923-937.
Representative United States patents that teach the preparation of
such oligonucleotide conjugates include, but are not limited to,
U.S. Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465;
5,541,313; 5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731;
5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603;
5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025;
4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582;
5,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963;
5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250;
5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463;
5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142;
5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928
and 5,688,941, certain of which are commonly owned with the instant
application, and each of which is herein incorporated by
reference.
It is not necessary for all positions in a given compound to be
uniformly modified, and in fact more than one of the aforementioned
modifications may be incorporated in a single compound or even at a
single nucleoside within an oligonucleotide. The present invention
also includes antisense compounds which are chimeric compounds.
"Chimeric" antisense compounds or "chimeras," in the context of
this invention, are antisense compounds, particularly
oligonucleotides, which contain two or more chemically distinct
regions, each made up of at least one monomer unit, i.e., a
nucleotide in the case of an oligonucleotide compound. These
oligonucleotides typically contain at least one region wherein the
oligonucleotide is modified so as to confer upon the
oligonucleotide increased resistance to nuclease degradation,
increased cellular uptake, and/or increased binding affinity for
the target nucleic acid. An additional region of the
oligonucleotide may serve as a substrate for enzymes capable of
cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is
a cellular endonuclease which cleaves the RNA strand of an RNA:DNA
duplex. Activation of RNase H, therefore, results in cleavage of
the RNA target, thereby greatly enhancing the efficiency of
oligonucleotide inhibition of gene expression. Consequently,
comparable results can often be obtained with shorter
oligonucleotides when chimeric oligonucleotides are used, compared
to phosphorothioate deoxyoligonucleotides hybridizing to the same
target region. Cleavage of the RNA target can be routinely detected
by gel electrophoresis and, if necessary, associated nucleic acid
hybridization techniques known in the art.
Chimeric antisense compounds of the invention may be formed as
composite structures of two or more oligonucleotides, modified
oligonucleotides, oligonucleosides and/or oligonucleotide mimetics
as described above. Such compounds have also been referred to in
the art as hybrids or gapmers. Representative United States patents
that teach the preparation of such hybrid structures include, but
are not limited to, U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007;
5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065;
5,652,355; 5,652,356; and 5,700,922, certain of which are commonly
owned with the instant application, and each of which is herein
incorporated by reference in its entirety.
The antisense compounds used in accordance with this invention may
be conveniently and routinely made through the well-known technique
of solid phase synthesis. Equipment for such synthesis is sold by
several vendors including, for example, Applied Biosystems (Foster
City, Calif.). Any other means for such synthesis known in the art
may additionally or alternatively be employed. It is well known to
use similar techniques to prepare oligonucleotides such as the
phosphorothioates and alkylated derivatives.
The antisense compounds of the invention are synthesized in vitro
and do not include antisense compositions of biological origin, or
genetic vector constructs designed to direct the in vivo synthesis
of antisense molecules. The compounds of the invention may also be
admixed, encapsulated, conjugated or otherwise associated with
other molecules, molecule structures or mixtures of compounds, as
for example, liposomes, receptor targeted molecules, oral, rectal,
topical or other formulations, for assisting in uptake,
distribution and/or absorption. Representative United States
patents that teach the preparation of such uptake, distribution
and/or absorption assisting formulations include, but are not
limited to, U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016;
5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721;
4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170;
5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854;
5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948;
5,580,575; and 5,595,756, each of which is herein incorporated by
reference.
The antisense compounds of the invention encompass any
pharmaceutically acceptable salts, esters, or salts of such esters,
or any other compound which, upon administration to an animal
including a human, is capable of providing (directly or indirectly)
the biologically active metabolite or residue thereof. Accordingly,
for example, the disclosure is also drawn to prodrugs and
pharmaceutically acceptable salts of the compounds of the
invention, pharmaceutically acceptable salts of such prodrugs, and
other bioequivalents.
The term "prodrug" indicates a therapeutic agent that is prepared
in an inactive form that is converted to an active form (i.e.,
drug) within the body or cells thereof by the action of endogenous
enzymes or other chemicals and/or conditions. In particular,
prodrug versions of the oligonucleotides of the invention are
prepared as SATE [(S-acetyl-2-thioethyl) phosphate] derivatives
according to the methods disclosed in WO 93/24510 to Gosselin et
al., published Dec. 9, 1993 or in WO 94/26764 to Imbach et al.
The term "pharmaceutically acceptable salts" refers to
physiologically and pharmaceutically acceptable salts of the
compounds of the invention: i.e., salts that retain the desired
biological activity of the parent compound and do not impart
undesired toxicological effects thereto.
Pharmaceutically acceptable base addition salts are formed with
metals or amines, such as alkali and alkaline earth metals or
organic amines. Examples of metals used as cations are sodium,
potassium, magnesium, calcium, and the like. Examples of suitable
amines are N,N'-dibenzylethylenediamine, chloroprocaine, choline,
diethanolamine, dicyclohexylamine, ethylenediamine,
N-methylglucamine, and procaine (see, for example, Berge et al.,
"Pharmaceutical Salts," J. of Pharma Sci., 1977, 66, 1-19). The
base addition salts of said acidic compounds are prepared by
contacting the free acid form with a sufficient amount of the
desired base to produce the salt in the conventional manner. The
free acid form may be regenerated by contacting the salt form with
an acid and isolating the free acid in the conventional manner. The
free acid forms differ from their respective salt forms somewhat in
certain physical properties such as solubility in polar solvents,
but otherwise the salts are equivalent to their respective free
acid for purposes of the present invention. As used herein, a
"pharmaceutical addition salt" includes a pharmaceutically
acceptable salt of an acid form of one of the components of the
compositions of the invention. These include organic or inorganic
acid salts of the amines. Preferred acid salts are the
hydrochlorides, acetates, salicylates, nitrates and phosphates.
Other suitable pharmaceutically acceptable salts are well known to
those skilled in the art and include basic salts of a variety of
inorganic and organic acids, such as, for example, with inorganic
acids, such as for example hydrochloric acid, hydrobromic acid,
sulfuric acid or phosphoric acid; with organic carboxylic,
sulfonic, sulfo or phospho acids or N-substituted sulfamic acids,
for example acetic acid, propionic acid, glycolic acid, succinic
acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric
acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic
acid, glucaric acid, glucuronic acid, citric acid, benzoic acid,
cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic
acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid,
nicotinic acid or isonicotinic acid; and with amino acids, such as
the 20 alpha-amino acids involved in the synthesis of proteins in
nature, for example glutamic acid or aspartic acid, and also with
phenylacetic acid, methanesulfonic acid, ethanesulfonic acid,
2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid,
benzenesulfonic acid, 4-methylbenzenesulfonic acid,
naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or
3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid
(with the formation of cyclamates), or with other acid organic
compounds, such as ascorbic acid. Pharmaceutically acceptable salts
of compounds may also be prepared with a pharmaceutically
acceptable cation. Suitable pharmaceutically acceptable cations are
well known to those skilled in the art and include alkaline,
alkaline earth, ammonium and quaternary ammonium cations.
Carbonates or hydrogen carbonates are also possible.
For oligonucleotides, preferred examples of pharmaceutically
acceptable salts include but are not limited to (a) salts formed
with cations such as sodium, potassium, ammonium, magnesium,
calcium, polyamines such as spermine and spermidine, etc.; (b) acid
addition salts formed with inorganic acids, for example
hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric
acid, nitric acid and the like; (c) salts formed with organic acids
such as, for example, acetic acid, oxalic acid, tartaric acid,
succinic acid, maleic acid, fumaric acid, gluconic acid, citric
acid, malic acid, ascorbic acid, benzoic acid, tannic acid,
palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic
acid, methanesulfonic acid, p-toluenesulfonic acid,
naphthalenedisulfonic acid, polygalacturonic acid, and the like;
and (d) salts formed from elemental anions such as chlorine,
bromine, and iodine.
The antisense compounds of the present invention can be utilized
for diagnostics, therapeutics, prophylaxis and as research reagents
and kits. For therapeutics, an animal, preferably a human,
suspected of having a disease or disorder which can be treated by
modulating the expression of integrin beta 3 is treated by
administering antisense compounds in accordance with this
invention. The compounds of the invention can be utilized in
pharmaceutical compositions by adding an effective amount of an
antisense compound to a suitable pharmaceutically acceptable
diluent or carrier. Use of the antisense compounds and methods of
the invention may also be useful prophylactically, e.g., to prevent
or delay infection, inflammation or tumor formation, for
example.
The antisense compounds of the invention are useful for research
and diagnostics, because these compounds hybridize to nucleic acids
encoding integrin beta 3, enabling sandwich and other assays to
easily be constructed to exploit this fact. Hybridization of the
antisense oligonucleotides of the invention with a nucleic acid
encoding integrin beta 3 can be detected by means known in the art.
Such means may include conjugation of an enzyme to the
oligonucleotide, radiolabelling of the oligonucleotide or any other
suitable detection means. Kits using such detection means for
detecting the level of integrin beta 3 in a sample may also be
prepared.
The present invention also includes pharmaceutical compositions and
formulations which include the antisense compounds of the
invention. The pharmaceutical compositions of the present invention
may be administered in a number of ways depending upon whether
local or systemic treatment is desired and upon the area to be
treated. Administration may be topical (including ophthalmic and to
mucous membranes including vaginal and rectal delivery), pulmonary,
e.g., by inhalation or insufflation of powders or aerosols,
including by nebulizer; intratracheal, intranasal, epidermal and
transdermal), oral or parenteral. Parenteral administration
includes intravenous, intraarterial, subcutaneous, intraperitoneal
or intramuscular injection or infusion; or intracranial, e.g.,
intrathecal or intraventricular, administration. Oligonucleotides
with at least one 2'-O-methoxyethyl modification are believed to be
particularly useful for oral administration.
Pharmaceutical compositions and formulations for topical
administration may include transdermal patches, ointments, lotions,
creams, gels, drops, suppositories, sprays, liquids and powders.
Conventional pharmaceutical carriers, aqueous, powder or oily
bases, thickeners and the like may be necessary or desirable.
Coated condoms, gloves and the like may also be useful.
Compositions and formulations for oral administration include
powders or granules, suspensions or solutions in water or
non-aqueous media, capsules, sachets or tablets. Thickeners,
flavoring agents, diluents, emulsifiers, dispersing aids or binders
may be desirable.
Compositions and formulations for parenteral, intrathecal or
intraventricular administration may include sterile aqueous
solutions which may also contain buffers, diluents and other
suitable additives such as, but not limited to, penetration
enhancers, carrier compounds and other pharmaceutically acceptable
carriers or excipients.
Pharmaceutical compositions of the present invention include, but
are not limited to, solutions, emulsions, and liposome-containing
formulations. These compositions may be generated from a variety of
components that include, but are not limited to, preformed liquids,
self-emulsifying solids and self-emulsifying semisolids.
The pharmaceutical formulations of the present invention, which may
conveniently be presented in unit dosage form, may be prepared
according to conventional techniques well known in the
pharmaceutical industry. Such techniques include the step of
bringing into association the active ingredients with the
pharmaceutical carrier(s) or excipient(s). In general the
formulations are prepared by uniformly and intimately bringing into
association the active ingredients with liquid carriers or finely
divided solid carriers or both, and then, if necessary, shaping the
product.
The compositions of the present invention may be formulated into
any of many possible dosage forms such as, but not limited to,
tablets, capsules, liquid syrups, soft gels, suppositories, and
enemas. The compositions of the present invention may also be
formulated as suspensions in aqueous, non-aqueous or mixed media.
Aqueous suspensions may further contain substances which increase
the viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers.
In one embodiment of the present invention the pharmaceutical
compositions may be formulated and used as foams. Pharmaceutical
foams include formulations such as, but not limited to, emulsions,
microemulsions, creams, jellies and liposomes. While basically
similar in nature these formulations vary in the components and the
consistency of the final product. The preparation of such
compositions and formulations is generally known to those skilled
in the pharmaceutical and formulation arts and may be applied to
the formulation of the compositions of the present invention.
Emulsions
The compositions of the present invention may be prepared and
formulated as emulsions. Emulsions are typically heterogenous
systems of one liquid dispersed in another in the form of droplets
usually exceeding 0.1 .mu.m in diameter. (Idson, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block
in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker
(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p.
335; Higuchi et al., in Remington's Pharmaceutical Sciences, Mack
Publishing Co., Easton, Pa., 1985, p. 301). Emulsions are often
biphasic systems comprising of two immiscible liquid phases
intimately mixed and dispersed with each other. In general,
emulsions may be either water-in-oil (w/o) or of the oil-in-water
(o/w) variety. When an aqueous phase is finely divided into and
dispersed as minute droplets into a bulk oily phase the resulting
composition is called a water-in-oil (w/o) emulsion. Alternatively,
when an oily phase is finely divided into and dispersed as minute
droplets into a bulk aqueous phase the resulting composition is
called an oil-in-water (o/w) emulsion. Emulsions may contain
additional components in addition to the dispersed phases and the
active drug which may be present as a solution in either the
aqueous phase, oily phase or itself as a separate phase.
Pharmaceutical excipients such as emulsifiers, stabilizers, dyes,
and anti-oxidants may also be present in emulsions as needed.
Pharmaceutical emulsions may also be multiple emulsions that are
comprised of more than two phases such as, for example, in the case
of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w)
emulsions. Such complex formulations often provide certain
advantages that simple binary emulsions do not. Multiple emulsions
in which individual oil droplets of an o/w emulsion enclose small
water droplets constitute a w/o/w emulsion. Likewise a system of
oil droplets enclosed in globules of water stabilized in an oily
continuous provides an o/w/o emulsion.
Emulsions are characterized by little or no thermodynamic
stability. Often, the dispersed or discontinuous phase of the
emulsion is well dispersed into the external or continuous phase
and maintained in this form through the means of emulsifiers or the
viscosity of the formulation. Either of the phases of the emulsion
may be a semisolid or a solid, as is the case of emulsion-style
ointment bases and creams. Other means of stabilizing emulsions
entail the use of emulsifiers that may be incorporated into either
phase of the emulsion. Emulsifiers may broadly be classified into
four categories: synthetic surfactants, naturally occurring
emulsifiers, absorption bases, and finely dispersed solids (Idson,
in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker
(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.
199).
Synthetic surfactants, also known as surface active agents, have
found wide applicability in the formulation of emulsions and have
been reviewed in the literature (Rieger, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 285; Idson, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), Marcel Dekker,
Inc., New York, N.Y., 1988, volume 1, p. 199). Surfactants are
typically amphiphilic and comprise a hydrophilic and a hydrophobic
portion. The ratio of the hydrophilic to the hydrophobic nature of
the surfactant has been termed the hydrophile/lipophile balance
(HLB) and is a valuable tool in categorizing and selecting
surfactants in the preparation of formulations. Surfactants may be
classified into different classes based on the nature of the
hydrophilic group: nonionic, anionic, cationic and amphoteric
(Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and
Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,
p. 285).
Naturally occurring emulsifiers used in emulsion formulations
include lanolin, beeswax, phosphatides, lecithin and acacia.
Absorption bases possess hydrophilic properties such that they can
soak up water to form w/o emulsions yet retain their semisolid
consistencies, such as anhydrous lanolin and hydrophilic
petrolatum. Finely divided solids have also been used as good
emulsifiers especially in combination with surfactants and in
viscous preparations. These include polar inorganic solids, such as
heavy metal hydroxides, nonswelling clays such as bentonite,
attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum
silicate and colloidal magnesium aluminum silicate, pigments and
nonpolar solids such as carbon or glyceryl tristearate.
A large variety of non-emulsifying materials are also included in
emulsion formulations and contribute to the properties of
emulsions. These include fats, oils, waxes, fatty acids, fatty
alcohols, fatty esters, humectants, hydrophilic colloids,
preservatives and antioxidants (Block, in Pharmaceutical Dosage
Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,
Inc., New York, N.Y., volume 1, p. 335; Idson, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 199).
Hydrophilic colloids or hydrocolloids include naturally occurring
gums and synthetic polymers such as polysaccharides (for example,
acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and
tragacanth), cellulose derivatives (for example,
carboxymethylcellulose and carboxypropylcellulose), and synthetic
polymers (for example, carbomers, cellulose ethers, and
carboxyvinyl polymers). These disperse or swell in water to form
colloidal solutions that stabilize emulsions by forming strong
interfacial films around the dispersed-phase droplets and by
increasing the viscosity of the external phase.
Since emulsions often contain a number of ingredients such as
carbohydrates, proteins, sterols and phosphatides that may readily
support the growth of microbes, these formulations often
incorporate preservatives. Commonly used preservatives included in
emulsion formulations include methyl paraben, propyl paraben,
quaternary ammonium salts, benzalkonium chloride, esters of
p-hydroxybenzoic acid, and boric acid. Antioxidants are also
commonly added to emulsion formulations to prevent deterioration of
the formulation. Antioxidants used may be free radical scavengers
such as tocopherols, alkyl gallates, butylated hydroxyanisole,
butylated hydroxytoluene, or reducing agents such as ascorbic acid
and sodium metabisulfite, and antioxidant synergists such as citric
acid, tartaric acid, and lecithin.
The application of emulsion formulations via dermatological, oral
and parenteral routes and methods for their manufacture have been
reviewed in the literature (Idson, in Pharmaceutical Dosage Forms,
Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New
York, N.Y., volume 1, p. 199). Emulsion formulations for oral
delivery have been very widely used because of reasons of ease of
formulation, efficacy from an absorption and bioavailability
standpoint. (Rosoff, in Pharmaceutical Dosage Forms, Lieberman,
Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York,
N.Y., volume 1, p. 245; Idson, in Pharmaceutical Dosage Forms,
Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New
York, N.Y., volume 1, p. 199). Mineral-oil base laxatives,
oil-soluble vitamins and high fat nutritive preparations are among
the materials that have commonly been administered orally as o/w
emulsions.
In one embodiment of the present invention, the compositions of
oligonucleotides and nucleic acids are formulated as
microemulsions. A microemulsion may be defined as a system of
water, oil and amphiphile which is a single optically isotropic and
thermodynamically stable liquid solution (Rosoff, in Pharmaceutical
Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel
Dekker, Inc., New York, N.Y., volume 1, p. 245). Typically
microemulsions are systems that are prepared by first dispersing an
oil in an aqueous surfactant solution and then adding a sufficient
amount of a fourth component, generally an intermediate
chain-length alcohol to form a transparent system. Therefore,
microemulsions have also been described as thermodynamically
stable, isotropically clear dispersions of two immiscible liquids
that are stabilized by interfacial films of surface-active
molecules (Leung and Shah, in: Controlled Release of Drugs:
Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH
Publishers, New York, pages 185-215). Microemulsions commonly are
prepared via a combination of three to five components that include
oil, water, surfactant, cosurfactant and electrolyte. Whether the
microemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w)
type is dependent on the properties of the oil and surfactant used
and on the structure and geometric packing of the polar heads and
hydrocarbon tails of the surfactant molecules (Schott, in
Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,
Pa., 1985, p. 271).
The phenomenological approach utilizing phase diagrams has been
extensively studied and has yielded a comprehensive knowledge, to
one skilled in the art, of how to formulate microemulsions (Rosoff,
in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker
(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p.
245; Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and
Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1,
p. 335). Compared to conventional emulsions, microemulsions offer
the advantage of solubilizing water-insoluble drugs in a
formulation of thermodynamically stable droplets that are formed
spontaneously.
Surfactants used in the preparation of microemulsions include, but
are not limited to, ionic surfactants, non-ionic surfactants, Brij
96, polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,
tetraglycerol monolaurate (ML310), tetraglycerol monooleate
(MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate
(PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate
(MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate
(DAO750), alone or in combination with cosurfactants. The
cosurfactant, usually a short-chain alcohol such as ethanol,
1-propanol, and 1-butanol, serves to increase the interfacial
fluidity by penetrating into the surfactant film and consequently
creating a disordered film because of the void space generated
among surfactant molecules. Microemulsions may, however, be
prepared without the use of cosurfactants and alcohol-free
self-emulsifying microemulsion systems are known in the art. The
aqueous phase may typically be, but is not limited to, water, an
aqueous solution of the drug, glycerol, PEG300, PEG400,
polyglycerols, propylene glycols, and derivatives of ethylene
glycol. The oil phase may include, but is not limited to, materials
such as Captex 300, Captex 355, Capmul MCM, fatty acid esters,
medium chain (C8-C12) mono, di, and triglycerides, polyoxyethylated
glyceryl fatty acid esters, fatty alcohols, polyglycolized
glycerides, saturated polyglycolized C8-C10 glycerides, vegetable
oils and silicone oil.
Microemulsions are particularly of interest from the standpoint of
drug solubilization and the enhanced absorption of drugs. Lipid
based microemulsions (both o/w and w/o) have been proposed to
enhance the oral bioavailability of drugs, including peptides
(Constantinides et al., Pharmaceutical Research, 1994, 11,
1385-1390; Ritschel, Meth. Find. Exp. Clin. Pharmacol., 1993, 13,
205). Microemulsions afford advantages of improved drug
solubilization, protection of drug from enzymatic hydrolysis,
possible enhancement of drug absorption due to surfactant-induced
alterations in membrane fluidity and permeability, ease of
preparation, ease of oral administration over solid dosage forms,
improved clinical potency, and decreased toxicity (Constantinides
et al., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J.
Pharm. Sci., 1996, 85, 138-143). Often microemulsions may form
spontaneously when their components are brought together at ambient
temperature. This may be particularly advantageous when formulating
thermolabile drugs, peptides or oligonucleotides. Microemulsions
have also been effective in the transdermal delivery of active
components in both cosmetic and pharmaceutical applications. It is
expected that the microemulsion compositions and formulations of
the present invention will facilitate the increased systemic
absorption of oligonucleotides and nucleic acids from the
gastrointestinal tract, as well as improve the local cellular
uptake of oligonucleotides and nucleic acids within the
gastrointestinal tract, vagina, buccal cavity and other areas of
administration.
Microemulsions of the present invention may also contain additional
components and additives such as sorbitan monostearate (Grill 3),
Labrasol, and penetration enhancers to improve the properties of
the formulation and to enhance the absorption of the
oligonucleotides and nucleic acids of the present invention.
Penetration enhancers used in the microemulsions of the present
invention may be classified as belonging to one of five broad
categories--surfactants, fatty acids, bile salts, chelating agents,
and non-chelating non-surfactants (Lee et al., Critical Reviews in
Therapeutic Drug Carrier Systems, 1991, p. 92). Each of these
classes has been discussed above.
Liposomes
There are many organized surfactant structures besides
microemulsions that have been studied and used for the formulation
of drugs. These include monolayers, micelles, bilayers and
vesicles. Vesicles, such as liposomes, have attracted great
interest because of their specificity and the duration of action
they offer from the standpoint of drug delivery. As used in the
present invention, the term "liposome" means a vesicle composed of
amphiphilic lipids arranged in a spherical bilayer or bilayers.
Liposomes are unilamellar or multilamellar vesicles which have a
membrane formed from a lipophilic material and an aqueous interior.
The aqueous portion contains the composition to be delivered.
Cationic liposomes possess the advantage of being able to fuse to
the cell wall. Non-cationic liposomes, although not able to fuse as
efficiently with the cell wall, are taken up by macrophages in
vivo.
In order to cross intact mammalian skin, lipid vesicles must pass
through a series of fine pores, each with a diameter less than 50
nm, under the influence of a suitable transdermal gradient.
Therefore, it is desirable to use a liposome which is highly
deformable and able to pass through such fine pores.
Further advantages of liposomes include; liposomes obtained from
natural phospholipids are biocompatible and biodegradable;
liposomes can incorporate a wide range of water and lipid soluble
drugs; liposomes can protect encapsulated drugs in their internal
compartments from metabolism and degradation (Rosoff, in
Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.),
1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245).
Important considerations in the preparation of liposome
formulations are the lipid surface charge, vesicle size and the
aqueous volume of the liposomes.
Liposomes are useful for the transfer and delivery of active
ingredients to the site of action. Because the liposomal membrane
is structurally similar to biological membranes, when liposomes are
applied to a tissue, the liposomes start to merge with the cellular
membranes. As the merging of the liposome and cell progresses, the
liposomal contents are emptied into the cell where the active agent
may act.
Liposomal formulations have been the focus of extensive
investigation as the mode of delivery for many drugs. There is
growing evidence that for topical administration, liposomes present
several advantages over other formulations. Such advantages include
reduced side-effects related to high systemic absorption of the
administered drug, increased accumulation of the administered drug
at the desired target, and the ability to administer a wide variety
of drugs, both hydrophilic and hydrophobic, into the skin.
Several reports have detailed the ability of liposomes to deliver
agents including high-molecular weight DNA into the skin. Compounds
including analgesics, antibodies, hormones and high-molecular
weight DNAs have been administered to the skin. The majority of
applications resulted in the targeting of the upper epidermis.
Liposomes fall into two broad classes. Cationic liposomes are
positively charged liposomes which interact with the negatively
charged DNA molecules to form a stable complex. The positively
charged DNA/liposome complex binds to the negatively charged cell
surface and is internalized in an endosome. Due to the acidic pH
within the endosome, the liposomes are ruptured, releasing their
contents into the cell cytoplasm (Wang et al., Biochem. Biophys.
Res. Commun., 1987, 147, 980-985).
Liposomes which are pH-sensitive or negatively-charged, entrap DNA
rather than complex with it. Since both the DNA and the lipid are
similarly charged, repulsion rather than complex formation occurs.
Nevertheless, some DNA is entrapped within the aqueous interior of
these liposomes. pH-sensitive liposomes have been used to deliver
DNA encoding the thymidine kinase gene to cell monolayers in
culture. Expression of the exogenous gene was detected in the
target cells (Zhou et al., Journal of Controlled Release, 1992, 19,
269-274).
One major type of liposomal composition includes phospholipids
other than naturally-derived phosphatidylcholine. Neutral liposome
compositions, for example, can be formed from dimyristoyl
phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine
(DPPC). Anionic liposome compositions generally are formed from
dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes
are formed primarily from dioleoyl phosphatidylethanolamine (DOPE).
Another type of liposomal composition is formed from
phosphatidylcholine (PC) such as, for example, soybean PC, and egg
PC. Another type is formed from mixtures of phospholipid and/or
phosphatidylcholine and/or cholesterol.
Several studies have assessed the topical delivery of liposomal
drug formulations to the skin. Application of liposomes containing
interferon to guinea pig skin resulted in a reduction of skin
herpes sores while delivery of interferon via other means (e.g. as
a solution or as an emulsion) were ineffective (Weiner et al.,
Journal of Drug Targeting, 1992, 2, 405-410). Further, an
additional study tested the efficacy of interferon administered as
part of a liposomal formulation to the administration of interferon
using an aqueous system, and concluded that the liposomal
formulation was superior to aqueous administration (du Plessis et
al., Antiviral Research, 1992, 18, 259-265).
Non-ionic liposomal systems have also been examined to determine
their utility in the delivery of drugs to the skin, in particular
systems comprising non-ionic surfactant and cholesterol. Non-ionic
liposomal formulations comprising Novasome.TM. I (glyceryl
dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and
Novasome.TM. II (glyceryl
distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used
to deliver cyclosporin-A into the dermis of mouse skin. Results
indicated that such non-ionic liposomal systems were effective in
facilitating the deposition of cyclosporin-A into different layers
of the skin (Hu et al. S.T.P. Pharma. Sci., 1994, 4, 6, 466).
Liposomes also include "sterically stabilized" liposomes, a term
which, as used herein, refers to liposomes comprising one or more
specialized lipids that, when incorporated into liposomes, result
in enhanced circulation lifetimes relative to liposomes lacking
such specialized lipids. Examples of sterically stabilized
liposomes are those in which part of the vesicle-forming lipid
portion of the liposome (A) comprises one or more glycolipids, such
as monosialoganglioside G.sub.M1, or (B) is derivatized with one or
more hydrophilic polymers, such as a polyethylene glycol (PEG)
moiety. While not wishing to be bound by any particular theory, it
is thought in the art that, at least for sterically stabilized
liposomes containing gangliosides, sphingomyelin, or
PEG-derivatized lipids, the enhanced circulation half-life of these
sterically stabilized liposomes derives from a reduced uptake into
cells of the reticuloendothelial system (RES) (Allen et al., FEBS
Letters, 1987, 223, 42; Wu et al., Cancer Research, 1993, 53,
3765).
Various liposomes comprising one or more glycolipids are known in
the art. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507,
64) reported the ability of monosialoganglioside G.sub.M1,
galactocerebroside sulfate and phosphatidylinositol to improve
blood half-lives of liposomes. These findings were expounded upon
by Gabizon et al. (Proc. Natl. Acad. Sci. U.S.A., 1988, 85, 6949).
U.S. Pat. No. 4,837,028 and WO 88/04924, both to Allen et al.,
disclose liposomes comprising (1) sphingomyelin and (2) the
ganglioside G.sub.M1 or a galactocerebroside sulfate ester. U.S.
Pat. No. 5,543,152 (Webb et al.) discloses liposomes comprising
sphingomyelin. Liposomes comprising
1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499
(Lim et al.).
Many liposomes comprising lipids derivatized with one or more
hydrophilic polymers, and methods of preparation thereof, are known
in the art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)
described liposomes comprising a nonionic detergent, 2C.sub.12 15G,
that contains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167,
79) noted that hydrophilic coating of polystyrene particles with
polymeric glycols results in significantly enhanced blood
half-lives. Synthetic phospholipids modified by the attachment of
carboxylic groups of polyalkylene glycols (e.g., PEG) are described
by Sears (U.S. Pat. Nos. 4,426,330 and 4,534,899). Klibanov et al.
(FEBS Lett., 1990, 268, 235) described experiments demonstrating
that liposomes comprising phosphatidylethanolamine (PE) derivatized
with PEG or PEG stearate have significant increases in blood
circulation half-lives. Blume et al. (Biochimica et Biophysica
Acta, 1990, 1029, 91) extended such observations to other
PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the
combination of distearoylphosphatidylethanolamine (DSPE) and PEG.
Liposomes having covalently bound PEG moieties on their external
surface are described in European Patent No. EP 0 445 131 B1 and WO
90/04384 to Fisher. Liposome compositions containing 1-20 mole
percent of PE derivatized with PEG, and methods of use thereof, are
described by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633)
and Martin et al. (U.S. Pat. No. 5,213,804 and European Patent No.
EP 0 496 813 B1). Liposomes comprising a number of other
lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat.
No. 5,225,212 (both to Martin et al.) and in WO 94/20073 (Zalipsky
et al.) Liposomes comprising PEG-modified ceramide lipids are
described in WO 96/10391 (Choi et al.). U.S. Pat. Nos. 5,540,935
(Miyazaki et al.) and 5,556,948 (Tagawa et al.) describe
PEG-containing liposomes that can be further derivatized with
functional moieties on their surfaces.
A limited number of liposomes comprising nucleic acids are known in
the art. WO 96/40062 to Thierry et al. discloses methods for
encapsulating high molecular weight nucleic acids in liposomes.
U.S. Pat. No. 5,264,221 to Tagawa et al. discloses protein-bonded
liposomes and asserts that the contents of such liposomes may
include an antisense RNA. U.S. Pat. No. 5,665,710 to Rahman et al.
describes certain methods of encapsulating oligodeoxynucleotides in
liposomes. WO 97/04787 to Love et al. discloses liposomes
comprising antisense oligonucleotides targeted to the raf gene.
Transfersomes are yet another type of liposomes, and are highly
deformable lipid aggregates which are attractive candidates for
drug delivery vehicles. Transfersomes may be described as lipid
droplets which are so highly deformable that they are easily able
to penetrate through pores which are smaller than the droplet.
Transfersomes are adaptable to the environment in which they are
used, e.g. they are self-optimizing (adaptive to the shape of pores
in the skin), self-repairing, frequently reach their targets
without fragmenting, and often self-loading. To make transfersomes
it is possible to add surface edge-activators, usually surfactants,
to a standard liposomal composition. Transfersomes have been used
to deliver serum albumin to the skin. The transfersome-mediated
delivery of serum albumin has been shown to be as effective as
subcutaneous injection of a solution containing serum albumin.
Surfactants find wide application in formulations such as emulsions
(including microemulsions) and liposomes. The most common way of
classifying and ranking the properties of the many different types
of surfactants, both natural and synthetic, is by the use of the
hydrophile/lipophile balance (HLB). The nature of the hydrophilic
group (also known as the "head") provides the most useful means for
categorizing the different surfactants used in formulations
(Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New
York, N.Y., 1988, p. 285).
If the surfactant molecule is not ionized, it is classified as a
nonionic surfactant. Nonionic surfactants find wide application in
pharmaceutical and cosmetic products and are usable over a wide
range of pH values. In general their HLB values range from 2 to
about 18 depending on their structure. Nonionic surfactants include
nonionic esters such as ethylene glycol esters, propylene glycol
esters, glyceryl esters, polyglyceryl esters, sorbitan esters,
sucrose esters, and ethoxylated esters. Nonionic alkanolamides and
ethers such as fatty alcohol ethoxylates, propoxylated alcohols,
and ethoxylated/propoxylated block polymers are also included in
this class. The polyoxyethylene surfactants are the most popular
members of the nonionic surfactant class.
If the surfactant molecule carries a negative charge when it is
dissolved or dispersed in water, the surfactant is classified as
anionic. Anionic surfactants include carboxylates such as soaps,
acyl lactylates, acyl amides of amino acids, esters of sulfuric
acid such as alkyl sulfates and ethoxylated alkyl sulfates,
sulfonates such as alkyl benzene sulfonates, acyl isethionates,
acyl taurates and sulfosuccinates, and phosphates. The most
important members of the anionic surfactant class are the alkyl
sulfates and the soaps.
If the surfactant molecule carries a positive charge when it is
dissolved or dispersed in water, the surfactant is classified as
cationic. Cationic surfactants include quaternary ammonium salts
and ethoxylated amines. The quaternary ammonium salts are the most
used members of this class.
If the surfactant molecule has the ability to carry either a
positive or negative charge, the surfactant is classified as
amphoteric. Amphoteric surfactants include acrylic acid
derivatives, substituted alkylamides, N-alkylbetaines and
phosphatides.
The use of surfactants in drug products, formulations and in
emulsions has been reviewed (Rieger, in Pharmaceutical Dosage
Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).
Penetration Enhancers
In one embodiment, the present invention employs various
penetration enhancers to effect the efficient delivery of nucleic
acids, particularly oligonucleotides, to the skin of animals. Most
drugs are present in solution in both ionized and nonionized forms.
However, usually only lipid soluble or lipophilic drugs readily
cross cell membranes. It has been discovered that even
non-lipophilic drugs may cross cell membranes if the membrane to be
crossed is treated with a penetration enhancer. In addition to
aiding the diffusion of non-lipophilic drugs across cell membranes,
penetration enhancers also enhance the permeability of lipophilic
drugs.
Penetration enhancers may be classified as belonging to one of five
broad categories, i.e., surfactants, fatty acids, bile salts,
chelating agents, and non-chelating non-surfactants (Lee et al.,
Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p.92).
Each of the above mentioned classes of penetration enhancers are
described below in greater detail.
Surfactants: In connection with the present invention, surfactants
(or "surface-active agents") are chemical entities which, when
dissolved in an aqueous solution, reduce the surface tension of the
solution or the interfacial tension between the aqueous solution
and another liquid, with the result that absorption of
oligonucleotides through the mucosa is enhanced. In addition to
bile salts and fatty acids, these penetration enhancers include,
for example, sodium lauryl sulfate, polyoxyethylene-9-lauryl ether
and polyoxyethylene-20-cetyl ether) (Lee et al., Critical Reviews
in Therapeutic Drug Carrier Systems, 1991, p.92); and
perfluorochemical emulsions, such as FC-43. Takahashi et al., J.
Pharm. Pharmacol., 1988, 40, 252).
Fatty acids: Various fatty acids and their derivatives which act as
penetration enhancers include, for example, oleic acid, lauric
acid, capric acid (n-decanoic acid), myristic acid, palmitic acid,
stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,
monoolein (1-monooleoyl-rac-glycerol), dilaurin, caprylic acid,
arachidonic acid, glycerol 1-monocaprate,
1-dodecylazacycloheptan-2-one, acylcarnitines, acylcholines,
C.sub.1-10 alkyl esters thereof (e.g., methyl, isopropyl and
t-butyl), and mono- and di-glycerides thereof (i.e., oleate,
laurate, caprate, myristate, palmitate, stearate, linoleate, etc.)
(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems,
1991, p.92; Muranishi, Critical Reviews in Therapeutic Drug Carrier
Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol.,
1992, 44, 651-654).
Bile salts: The physiological role of bile includes the
facilitation of dispersion and absorption of lipids and fat-soluble
vitamins (Brunton, Chapter 38 in: Goodman & Gilman's The
Pharmacological Basis of Therapeutics, 9th Ed., Hardman et al.
Eds., McGraw-Hill, New York, 1996, pp. 934-935). Various natural
bile salts, and their synthetic derivatives, act as penetration
enhancers. Thus the term "bile salts" includes any of the naturally
occurring components of bile as well as any of their synthetic
derivatives. The bile salts of the invention include, for example,
cholic acid (or its pharmaceutically acceptable sodium salt, sodium
cholate), dehydrocholic acid (sodium dehydrocholate), deoxycholic
acid (sodium deoxycholate), glucholic acid (sodium glucholate),
glycholic acid (sodium glycocholate), glycodeoxycholic acid (sodium
glycodeoxycholate), taurocholic acid (sodium taurocholate),
taurodeoxycholic acid (sodium taurodeoxycholate), chenodeoxycholic
acid (sodium chenodeoxycholate), ursodeoxycholic acid (UDCA),
sodium tauro-24,25-dihydro-fusidate (STDHF), sodium
glycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (Lee
et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991,
page 92; Swinyard, Chapter 39 In: Remington's Pharmaceutical
Sciences, 18th Ed., Gennaro, ed., Mack Publishing Co., Easton, Pa.,
1990, pages 782-783; Muranishi, Critical Reviews in Therapeutic
Drug Carrier Systems, 1990, 7, 1-33; Yamamoto et al., J. Pharm.
Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm. Sci., 1990,
79, 579-583).
Chelating Agents: Chelating agents, as used in connection with the
present invention, can be defined as compounds that remove metallic
ions from solution by forming complexes therewith, with the result
that absorption of oligonucleotides through the mucosa is enhanced.
With regards to their use as penetration enhancers in the present
invention, chelating agents have the added advantage of also
serving as DNase inhibitors, as most characterized DNA nucleases
require a divalent metal ion for catalysis and are thus inhibited
by chelating agents (Jarrett, J. Chromatogr., 1993, 618, 315-339).
Chelating agents of the invention include but are not limited to
disodium ethylenediaminetetraacetate (EDTA), citric acid,
salicylates (e.g., sodium salicylate, 5-methoxysalicylate and
homovanilate), N-acyl derivatives of collagen, laureth-9 and
N-amino acyl derivatives of beta-diketones (enamines) (Lee et al.,
Critical Reviews in Therapeutic Drug Carrier Systems, 1991, page
92; Muranishi, Critical Reviews in Therapeutic Drug Carrier
Systems, 1990, 7, 1-33; Buur et al., J. Control Rel., 1990, 14,
43-51).
Non-chelating non-surfactants: As used herein, non-chelating
non-surfactant penetration enhancing compounds can be defined as
compounds that demonstrate insignificant activity as chelating
agents or as surfactants but that nonetheless enhance absorption of
oligonucleotides through the alimentary mucosa (Muranishi, Critical
Reviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33). This
class of penetration enhancers include, for example, unsaturated
cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanone derivatives
(Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems,
1991, page 92); and non-steroidal anti-inflammatory agents such as
diclofenac sodium, indomethacin and phenylbutazone (Yamashita et
al., J. Pharm. Pharmacol., 1987, 39, 621-626).
Agents that enhance uptake of oligonucleotides at the cellular
level may also be added to the pharmaceutical and other
compositions of the present invention. For example, cationic
lipids, such as lipofectin (Junichi et al, U.S. Pat. No.
5,705,188), cationic glycerol derivatives, and polycationic
molecules, such as polylysine (Lollo et al., PCT Application WO
97/30731), are also known to enhance the cellular uptake of
oligonucleotides.
Other agents may be utilized to enhance the penetration of the
administered nucleic acids, including glycols such as ethylene
glycol and propylene glycol, pyrrols such as 2-pyrrol, azones, and
terpenes such as limonene and menthone.
Carriers
Certain compositions of the present invention also incorporate
carrier compounds in the formulation. As used herein, "carrier
compound" or "carrier" can refer to a nucleic acid, or analog
thereof, which is inert (i.e., does not possess biological activity
per se) but is recognized as a nucleic acid by in vivo processes
that reduce the bioavailability of a nucleic acid having biological
activity by, for example, degrading the biologically active nucleic
acid or promoting its removal from circulation. The
coadministration of a nucleic acid and a carrier compound,
typically with an excess of the latter substance, can result in a
substantial reduction of the amount of nucleic acid recovered in
the liver, kidney or other extracirculatory reservoirs, presumably
due to competition between the carrier compound and the nucleic
acid for a common receptor. For example, the recovery of a
partially phosphorothioate oligonucleotide in hepatic tissue can be
reduced when it is coadministered with polyinosinic acid, dextran
sulfate, polycytidic acid or
4-acetamido-4'isothiocyano-stilbene-2,2'-disulfonic acid (Miyao et
al., Antisense Res. Dev., 1995, 5, 115-121; Takakura et al.,
Antisense & Nucl. Acid Drug Dev., 1996, 6, 177-183).
Excipients
In contrast to a carrier compound, a "pharmaceutical carrier" or
"excipient" is a pharmaceutically acceptable solvent, suspending
agent or any other pharmacologically inert vehicle for delivering
one or more nucleic acids to an animal. The excipient may be liquid
or solid and is selected, with the planned manner of administration
in mind, so as to provide for the desired bulk, consistency, etc.,
when combined with a nucleic acid and the other components of a
given pharmaceutical composition. Typical pharmaceutical carriers
include, but are not limited to, binding agents (e.g.,
pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl
methylcellulose, etc.); fillers (e.g., lactose and other sugars,
microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl
cellulose, polyacrylates or calcium hydrogen phosphate, etc.);
lubricants (e.g., magnesium stearate, talc, silica, colloidal
silicon dioxide, stearic acid, metallic stearates, hydrogenated
vegetable oils, corn starch, polyethylene glycols, sodium benzoate,
sodium acetate, etc.); disintegrants (e.g., starch, sodium starch
glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate,
etc.).
Pharmaceutically acceptable organic or inorganic excipient suitable
for non-parenteral administration which do not deleteriously react
with nucleic acids can also be used to formulate the compositions
of the present invention. Suitable pharmaceutically acceptable
carriers include, but are not limited to, water, salt solutions,
alcohols, polyethylene glycols, gelatin, lactose, amylose,
magnesium stearate, talc, silicic acid, viscous paraffin,
hydroxymethylcellulose, polyvinylpyrrolidone and the like.
Formulations for topical administration of nucleic acids may
include sterile and non-sterile aqueous solutions, non-aqueous
solutions in common solvents such as alcohols, or solutions of the
nucleic acids in liquid or solid oil bases. The solutions may also
contain buffers, diluents and other suitable additives.
Pharmaceutically acceptable organic or inorganic excipients
suitable for non-parenteral administration which do not
deleteriously react with nucleic acids can be used.
Suitable pharmaceutically acceptable excipients include, but are
not limited to, water, salt solutions, alcohol, polyethylene
glycols, gelatin, lactose, amylose, magnesium stearate, talc,
silicic acid, viscous paraffin, hydroxymethylcellulose,
polyvinylpyrrolidone and the like.
Other Components
The compositions of the present invention may additionally contain
other adjunct components conventionally found in pharmaceutical
compositions, at their art-established usage levels. Thus, for
example, the compositions may contain additional, compatible,
pharmaceutically-active materials such as, for example,
antipruritics, astringents, local anesthetics or anti-inflammatory
agents, or may contain additional materials useful in physically
formulating various dosage forms of the compositions of the present
invention, such as dyes, flavoring agents, preservatives,
antioxidants, opacifiers, thickening agents and stabilizers.
However, such materials, when added, should not unduly interfere
with the biological activities of the components of the
compositions of the present invention. The formulations can be
sterilized and, if desired, mixed with auxiliary agents, e.g.,
lubricants, preservatives, stabilizers, wetting agents,
emulsifiers, salts for influencing osmotic pressure, buffers,
colorings, flavorings and/or aromatic substances and the like which
do not deleteriously interact with the nucleic acid(s) of the
formulation.
Aqueous suspensions may contain substances which increase the
viscosity of the suspension including, for example, sodium
carboxymethylcellulose, sorbitol and/or dextran. The suspension may
also contain stabilizers.
Certain embodiments of the invention provide pharmaceutical
compositions containing (a) one or more antisense compounds and (b)
one or more other chemotherapeutic agents which function by a
non-antisense mechanism. Examples of such chemotherapeutic agents
include, but are not limited to, anticancer drugs such as
daunorubicin, dactinomycin, doxorubicin, bleomycin, mitomycin,
nitrogen mustard, chlorambucil, melphalan, cyclophosphamide,
6-mercaptopurine, 6-thioguanine, cytarabine (CA), 5-fluorouracil
(5-FU), floxuridine (5-FUdR), methotrexate (MTX), colchicine,
vincristine, vinblastine, etoposide, teniposide, cisplatin and
diethylstilbestrol (DES). See, generally, The Merck Manual of
Diagnosis and Therapy, 15th Ed., Berkow et al., eds., 1987, Rahway,
N.J., pages 1206-1228). Anti-inflammatory drugs, including but not
limited to nonsteroidal anti-inflammatory drugs and
corticosteroids, and antiviral drugs, including but not limited to
ribivirin, vidarabine, acyclovir and ganciclovir, may also be
combined in compositions of the invention. See, generally, The
Merck Manual of Diagnosis and Therapy, 15th Ed., Berkow et al.,
eds., 1987, Rahway, N.J., pages 2499-2506 and 46-49, respectively).
Other non-antisense chemotherapeutic agents are also within the
scope of this invention. Two or more combined compounds may be used
together or sequentially.
In another related embodiment, compositions of the invention may
contain one or more antisense compounds, particularly
oligonucleotides, targeted to a first nucleic acid and one or more
additional antisense compounds targeted to a second nucleic acid
target. Numerous examples of antisense compounds are known in the
art. Two or more combined compounds may be used together or
sequentially.
The formulation of therapeutic compositions and their subsequent
administration is believed to be within the skill of those in the
art. Dosing is dependent on severity and responsiveness of the
disease state to be treated, with the course of treatment lasting
from several days to several months, or until a cure is effected or
a diminution of the disease state is achieved. Optimal dosing
schedules can be calculated from measurements of drug accumulation
in the body of the patient. Persons of ordinary skill can easily
determine optimum dosages, dosing methodologies and repetition
rates. Optimum dosages may vary depending on the relative potency
of individual oligonucleotides, and can generally be estimated
based on EC.sub.50 s found to be effective in in vitro and in vivo
animal models. In general, dosage is from 0.01 ug to 100 g per kg
of body weight, and may be given once or more daily, weekly,
monthly or yearly, or even once every 2 to 20 years. Persons of
ordinary skill in the art can easily estimate repetition rates for
dosing based on measured residence times and concentrations of the
drug in bodily fluids or tissues. Following successful treatment,
it may be desirable to have the patient undergo maintenance therapy
to prevent the recurrence of the disease state, wherein the
oligonucleotide is administered in maintenance doses, ranging from
0.01 ug to 100 g per kg of body weight, once or more daily, to once
every 20 years.
While the present invention has been described with specificity in
accordance with certain of its preferred embodiments, the following
examples serve only to illustrate the invention and are not
intended to limit the same.
EXAMPLES
Example 1
Nucleoside Phosphoramidites for Oligonucleotide Synthesis Deoxy and
2'-alkoxy Amidites
2'-Deoxy and 2'-methoxy beta-cyanoethyldiisopropyl phosphoramidites
were purchased from commercial sources (e.g. Chemgenes, Needham,
Mass. or Glen Research, Inc. Sterling, Va.). Other 2'-O-alkoxy
substituted nucleoside amidites are prepared as described in U.S.
Pat. No. 5,506,351, herein incorporated by reference. For
oligonucleotides synthesized using 2'-alkoxy amidites, the standard
cycle for unmodified oligonucleotides was utilized, except the wait
step after pulse delivery of tetrazole and base was increased to
360 seconds.
Oligonucleotides containing 5-methyl-2'-deoxycytidine (5-Me-C)
nucleotides were synthesized according to published methods
[Sanghvi, et. al., Nucleic Acids Research, 1993, 21, 3197-3203]
using commercially available phosphoramidites (Glen Research,
Sterling, Va. or ChemGenes, Needham, Mass.).
2'-Fluoro Amidites
2'-Fluorodeoxyadenosine amidites
2'-fluoro oligonucleotides were synthesized as described previously
[Kawasaki, et. al., J. Med. Chem., 1993, 36, 831-841] and U.S. Pat.
No. 5,670,633, herein incorporated by reference. Briefly, the
protected nucleoside N6-benzoyl-2'-deoxy-2'-fluoroadenosine was
synthesized utilizing commercially available
9-beta-D-arabinofuranosyladenine as starting material and by
modifying literature procedures whereby the 2'-alpha-fluoro atom is
introduced by a S.sub.N 2-displacement of a 2'-beta-trityl group.
Thus N6-benzoyl-9-beta-D-arabinofuranosyladenine was selectively
protected in moderate yield as the 3',5'-ditetrahydropyranyl (THP)
intermediate. Deprotection of the THP and N6-benzoyl groups was
accomplished using standard methodologies and standard methods were
used to obtain the 5'-dimethoxytrityl-(DMT) and
5'-DMT-3'-phosphoramidite intermediates.
2'-Fluorodeoxyguanosine
The synthesis of 2'-deoxy-2'-fluoroguanosine was accomplished using
tetraisopropyldisiloxanyl (TPDS) protected
9-beta-D-arabinofuranosylguanine as starting material, and
conversion to the intermediate
diisobutyryl-arabinofuranosylguanosine. Deprotection of the TPDS
group was followed by protection of the hydroxyl group with THP to
give diisobutyryl di-THP protected arabinofuranosylguanine.
Selective O-deacylation and triflation was followed by treatment of
the crude product with fluoride, then deprotection of the THP
groups. Standard methodologies were used to obtain the 5'-DMT- and
5'-DMT-3'-phosphoramidites.
2'-Fluorouridine
Synthesis of 2'-deoxy-2'-fluorouridine was accomplished by the
modification of a literature procedure in which
2,2'-anhydro-1-beta-D-arabinofuranosyluracil was treated with 70%
hydrogen fluoride-pyridine. Standard procedures were used to obtain
the 5'-DMT and 5'-DMT-3'phosphoramidites.
2'-Fluorodeoxycytidine
2'-deoxy-2'-fluorocytidine was synthesized via amination of
2'-deoxy-2'-fluorouridine, followed by selective protection to give
N4-benzoyl-2'-deoxy-2'-fluorocytidine. Standard procedures were
used to obtain the 5'-DMT and 5'-DMT-3-phosphoramidites.
2'-O-(2-Methoxyethyl) modified amidites
2'-O-Methoxyethyl-substituted nucleoside amidites are prepared as
follows, or alternatively, as per the methods of Martin, P.,
Helvetica Chimica Acta, 1995, 78, 486-504.
2,2'-Anhydro [1-(beta-D-arabinofuranosyl)-5-methyluridine]
5-Methyluridine (ribosylthymine, commercially available through
Yamasa, Choshi, Japan) (72.0 g, 0.279M), diphenyl-carbonate (90.0
g, 0.420M) and sodium bicarbonate (2.0 g, 0.024M) were added to DMF
(300 mL). The mixture was heated to reflux, with stirring, allowing
the evolved carbon dioxide gas to be released in a controlled
manner. After 1 hour, the slightly darkened solution was
concentrated under reduced pressure. The resulting syrup was poured
into diethylether (2.5 L), with stirring. The product formed a gum.
The ether was decanted and the residue was dissolved in a minimum
amount of methanol (ca. 400 mL). The solution was poured into fresh
ether (2.5 L) to yield a stiff gum. The ether was decanted and the
gum was dried in a vacuum oven (60.degree. C. at 1 mm Hg for 24 h)
to give a solid that was crushed to a light tan powder (57 g, 85%
crude yield). The NMR spectrum was consistent with the structure,
contaminated with phenol as its sodium salt (ca. 5%). The material
was used as is for further reactions (or it can be purified further
by column chromatography using a gradient of methanol in ethyl
acetate (10-25%) to give a white solid, mp 222-4.degree. C.).
2'-O-Methoxyethyl-5-methyluridine
2,2'-Anhydro-5-methyluridine (195 g, 0.81M),
tris(2-methoxyethyl)borate (231 g, 0.98M) and 2-methoxyethanol (1.2
L) were added to a 2 L stainless steel pressure vessel and placed
in a pre-heated oil bath at 160.degree. C. After heating for 48
hours at 155-160.degree. C., the vessel was opened and the solution
evaporated to dryness and triturated with MeOH (200 mL). The
residue was suspended in hot acetone (1 L). The insoluble salts
were filtered, washed with acetone (150 mL) and the filtrate
evaporated. The residue (280 g) was dissolved in CH.sub.3 CN (600
mL) and evaporated. A silica gel column (3 kg) was packed in
CH.sub.2 Cl.sub.2 /acetone/MeOH (20:5:3) containing 0.5% Et.sub.3
NH. The residue was dissolved in CH.sub.2 Cl.sub.2 (250 mL) and
adsorbed onto silica (150 g) prior to loading onto the column. The
product was eluted with the packing solvent to give 160 g (63%) of
product. Additional material was obtained by reworking impure
fractions.
2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine
2'-O-Methoxyethyl-5-methyluridine (160 g, 0.506M) was co-evaporated
with pyridine (250 mL) and the dried residue dissolved in pyridine
(1.3 L). A first aliquot of dimethoxytrityl chloride (94.3 g,
0.278M) was added and the mixture stirred at room temperature for
one hour. A second aliquot of dimethoxytrityl chloride (94.3 g,
0.278M) was added and the reaction stirred for an additional one
hour. Methanol (170 mL) was then added to stop the reaction. HPLC
showed the presence of approximately 70% product. The solvent was
evaporated and triturated with CH.sub.3 CN (200 mL). The residue
was dissolved in CHCl.sub.3 (1.5 L) and extracted with 2.times.500
mL of saturated NaHCO.sub.3 and 2.times.500 mL of saturated NaCl.
The organic phase was dried over Na.sub.2 SO.sub.4, filtered and
evaporated. 275 g of residue was obtained. The residue was purified
on a 3.5 kg silica gel column, packed and eluted with
EtOAc/hexane/acetone (5:5:1) containing 0.5% Et.sub.3 NH. The pure
fractions were evaporated to give 164 g of product. Approximately
20 g additional was obtained from the impure fractions to give a
total yield of 183 g (57%).
3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine
2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine (106 g,
0.167M), DMF/pyridine (750 mL of a 3:1 mixture prepared from 562 mL
of DMF and 188 mL of pyridine) and acetic anhydride (24.38 mL,
0.258M) were combined and stirred at room temperature for 24 hours.
The reaction was monitored by TLC by first quenching the TLC sample
with the addition of MeOH. Upon completion of the reaction, as
judged by TLC, MeOH (50 mL) was added and the mixture evaporated at
35.degree. C. The residue was dissolved in CHCl.sub.3 (800 mL) and
extracted with 2.times.200 mL of saturated sodium bicarbonate and
2.times.200 mL of saturated NaCl. The water layers were back
extracted with 200 mL of CHCl.sub.3. The combined organics were
dried with sodium sulfate and evaporated to give 122 g of residue
(approx. 90% product). The residue was purified on a 3.5 kg silica
gel column and eluted using EtOAc/hexane(4:1). Pure product
fractions were evaporated to yield 96 g (84%). An additional 1.5 g
was recovered from later fractions.
3'-O-Acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triazoleuridi
ne
A first solution was prepared by dissolving
3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyluridine
(96 g, 0.144M) in CH.sub.3 CN (700 mL) and set aside. Triethylamine
(189 mL, 1.44M) was added to a solution of triazole (90 g, 1.3M) in
CH.sub.3 CN (1 L), cooled to -5.degree. C. and stirred for 0.5 h
using an overhead stirrer. POCl.sub.3 was added dropwise, over a 30
minute period, to the stirred solution maintained at 0-10.degree.
C., and the resulting mixture stirred for an additional 2 hours.
The first solution was added dropwise, over a 45 minute period, to
the latter solution. The resulting reaction mixture was stored
overnight in a cold room. Salts were filtered from the reaction
mixture and the solution was evaporated. The residue was dissolved
in EtOAc (1 L) and the insoluble solids were removed by filtration.
The filtrate was washed with 1.times.300 mL of NaHCO.sub.3 and
2.times.300 mL of saturated NaCl, dried over sodium sulfate and
evaporated. The residue was triturated with EtOAc to give the title
compound.
2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine
A solution of
3'-O-acetyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methyl-4-triazoleurid
ine (103 g, 0.141M) in dioxane (500 mL) and NH.sub.4 OH (30 mL) was
stirred at room temperature for 2 hours. The dioxane solution was
evaporated and the residue azeotroped with MeOH (2.times.200 mL).
The residue was dissolved in MeOH (300 mL) and transferred to a 2
liter stainless steel pressure vessel. MeOH (400 mL) saturated with
NH.sub.3 gas was added and the vessel heated to 100.degree. C. for
2 hours (TLC showed complete conversion). The vessel contents were
evaporated to dryness and the residue was dissolved in EtOAc (500
mL) and washed once with saturated NaCl (200 mL). The organics were
dried over sodium sulfate and the solvent was evaporated to give 85
g (95%) of the title compound.
N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine
2'-O-Methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine (85 g,
0.134M) was dissolved in DMF (800 mL) and benzoic anhydride (37.2
g, 0.165M) was added with stirring. After stirring for 3 hours, TLC
showed the reaction to be approximately 95% complete. The solvent
was evaporated and the residue azeotroped with MeOH (200 mL). The
residue was dissolved in CHCl (700 mL) and extracted with saturated
NaHCO, (2.times.300 mL) and saturated NaCl (2.times.300 mL), dried
over MgSO.sub.4 and evaporated to give a residue (96 g). The
residue was chromatographed on a 1.5 kg silica column using
EtOAc/hexane (1:1) containing 0.5% Et.sub.3 NH as the eluting
solvent. The pure product fractions were evaporated to give 90 g
(90%) of the title compound.
N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine-3'-amidi
te
N4-Benzoyl-2'-O-methoxyethyl-5'-O-dimethoxytrityl-5-methylcytidine
(74 g, 0.10M) was dissolved in CH.sub.2 Cl.sub.2 (1 L). Tetrazole
diisopropylamine (7.1 g) and
2-cyanoethoxy-tetra-(isopropyl)phosphite (40.5 mL, 0.123M) were
added with stirring, under a nitrogen atmosphere. The resulting
mixture was stirred for 20 hours at room temperature (TLC showed
the reaction to be 95% complete). The reaction mixture was
extracted with saturated NaHCO.sub.3 (1.times.300 mL) and saturated
NaCl (3.times.300 mL). The aqueous washes were back-extracted with
CH.sub.2 Cl.sub.2 (300 mL), and the extracts were combined, dried
over MgSO.sub.4 and concentrated. The residue obtained was
chromatographed on a 1.5 kg silica column using EtOAc/hexane (3:1)
as the eluting solvent. The pure fractions were combined to give
90.6 g (87%) of the title compound.
2'-O-(Aminooxyethyl) Nucleoside Amidites and
2'-O-(dimethylaminooxyethyl) Nucleoside Amidites
2'-(Dimethylaminooxyethoxy) nucleoside amidites
2'-(Dimethylaminooxyethoxy) nucleoside amidites [also known in the
art as 2'-O-(dimethylaminooxyethyl) nucleoside amidites] are
prepared as described in the following paragraphs. Adenosine,
cytidine and guanosine nucleoside amidites are prepared similarly
to the thymidine (5-methyluridine) except the exocyclic amines are
protected with a benzoyl moiety in the case of adenosine and
cytidine and with isobutyryl in the case of guanosine.
5'-O-tert-Butyldiphenylsilyl-O.sup.2
-2'-anhydro-5-methyluridine
O.sup.2 -2'-anhydro-5-methyluridine (Pro. Bio. Sint., Varese,
Italy, 100.0 g, 0.416 mmol), dimethylaminopyridine (0.66 g, 0.013
eq, 0.0054 mmol) were dissolved in dry pyridine (500 ml) at ambient
temperature under an argon atmosphere and with mechanical stirring.
tert-Butyldiphenylchlorosilane (125.8 g, 119.0 mL, 1.1 eq, 0.458
mmol) was added in one portion. The reaction was stirred for 16 h
at ambient temperature. TLC (Rf 0.22, ethyl acetate) indicated a
complete reaction. The solution was concentrated under reduced
pressure to a thick oil. This was partitioned between
dichloromethane (1 L) and saturated sodium bicarbonate (2.times.1
L) and brine (1 L). The organic layer was dried over sodium sulfate
and concentrated under reduced pressure to a thick oil. The oil was
dissolved in a 1:1 mixture of ethyl acetate and ethyl ether (600
mL) and the solution was cooled to -10.degree. C. The resulting
crystalline product was collected by filtration, washed with ethyl
ether (3.times.200 mL) and dried (40.degree. C., 1 mm Hg, 24 h) to
149 g (74.8%) of white solid. TLC and NMR were consistent with pure
product.
5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine
In a 2 L stainless steel, unstirred pressure reactor was added
borane in tetrahydrofuran (1.0M, 2.0 eq, 622 mL). In the fume hood
and with manual stirring, ethylene glycol (350 mL, excess) was
added cautiously at first until the evolution of hydrogen gas
subsided. 5'-O-tert-Butyldiphenylsilyl-O.sub.2
-2'-anhydro-5-methyluridine (149 g, 0.311 mol) and sodium
bicarbonate (0.074 g, 0.003 eq) were added with manual stirring.
The reactor was sealed and heated in an oil bath until an internal
temperature of 160.degree. C. was reached and then maintained for
16 h (pressure<100 psig). The reaction vessel was cooled to
ambient and opened. TLC (Rf 0.67 for desired product and Rf 0.82
for ara-T side product, ethyl acetate) indicated about 70%
conversion to the product. In order to avoid additional side
product formation, the reaction was stopped, concentrated under
reduced pressure (10 to 1 mm Hg) in a warm water bath
(40-100.degree. C.) with the more extreme conditions used to remove
the ethylene glycol. [Alternatively, once the low boiling solvent
is gone, the remaining solution can be partitioned between ethyl
acetate and water. The product will be in the organic phase.] The
residue was purified by column chromatography (2 kg silica gel,
ethyl acetate-hexanes gradient 1:1 to 4:1). The appropriate
fractions were combined, stripped and dried to product as a white
crisp foam (84 g, 50%), contaminated starting material (17.4 g) and
pure reusable starting material 20 g. The yield based on starting
material less pure recovered starting material was 58%. TLC and NMR
were consistent with 99% pure product.
2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridine
5'-O-tert-Butyldiphenylsilyl-2'-O-(2-hydroxyethyl)-5-methyluridine
(20 g, 36.98 mmol) was mixed with triphenylphosphine (11.63 g,
44.36 mmol) and N-hydroxyphthalimide (7.24 g, 44.36 mmol). It was
then dried over P.sub.2 O.sub.5 under high vacuum for two days at
40.degree. C. The reaction mixture was flushed with argon and dry
THF (369.8 mL, Aldrich, sure seal bottle) was added to get a clear
solution. Diethyl-azodicarboxylate (6.98 mL, 44.36 mmol) was added
dropwise to the reaction mixture. The rate of addition is
maintained such that resulting deep red coloration is just
discharged before adding the next drop. After the addition was
complete, the reaction was stirred for 4 hrs. By that time TLC
showed the completion of the reaction (ethylacetate:hexane, 60:40).
The solvent was evaporated in vacuum. Residue obtained was placed
on a flash column and eluted with ethyl acetate:hexane (60:40), to
get
2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridine
as white foam (21.819 g, 86%).
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-methyluridi
ne
2'-O-([2-phthalimidoxy)ethyl]-5'-t-butyldiphenylsilyl-5-methyluridine
(3.1 g, 4.5 mmol) was dissolved in dry CH.sub.2 Cl.sub.2 (4.5 mL)
and methylhydrazine (300 mL, 4.64 mmol) was added dropwise at
-10.degree. C. to 0.degree. C. After 1 h the mixture was filtered,
the filtrate was washed with ice cold CH.sub.2 CL.sub.2 and the
combined organic phase was washed with water, brine and dried over
anhydrous Na.sub.2 SO.sub.4. The solution was concentrated to get
2'-O-(aminooxyethyl) thymidine, which was then dissolved in NeOH
(67.5 mL). To this formaldehyde (20% aqueous solution, w/w, 1.1
eq.) was added and the resulting mixture was strirred for 1 h.
Solvent was removed under vacuum; residue chromatographed to get
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)
ethyl]-5-methyluridine as white foam (1.95 g, 78%).
5'-O-tert-Butyldiphenylsilyl-2'-O-[N,N-dimethylaminooxyethyl]-5-methyluridi
ne
5'-O-tert-butyldiphenylsilyl-2'-O-[(2-formadoximinooxy)ethyl]-5-methyluridi
ne (1.77 g, 3.12 mmol) was dissolved in a solution of 1M pyridinium
p-toluenesulfonate (PPTS) in dry MeOH (30.6 mL). Sodium
cyanoborohydride (0.39 g, 6.13 mmol) was added to this solution at
10.degree. C. under inert atmosphere. The reaction mixture was
stirred for 10 minutes at 10.degree. C. After that the reaction
vessel was removed from the ice bath and stirred at room
temperature for 2 h, the reaction monitored by TLC (5% MeOH in
CH.sub.2 Cl.sub.2). Aqueous NaHCO.sub.3 solution (5%, 10 mL) was
added and extracted with ethyl acetate (2.times.20 mL). Ethyl
acetate phase was dried over anhydrous Na.sub.2 SO.sub.4,
evaporated to dryness. Residue was dissolved in a solution of 1M
PPTS in MeOH (30.6 mL). Formaldehyde (20% w/w, 30 mL, 3.37 mmol)
was added and the reaction mixture was stirred at room temperature
for 10 minutes. Reaction mixture cooled to 10.degree. C. in an ice
bath, sodium cyanoborohydride (0.39 g, 6.13 mmol) was added and
reaction mixture stirred at 10.degree. C. for 10 minutes. After 10
minutes, the reaction mixture was removed from the ice bath and
stirred at room temperature for 2 hrs. To the reaction mixture 5%
NaHCO.sub.3 (25 mL) solution was added and extracted with ethyl
acetate (2.times.25 mL). Ethyl acetate layer was dried over
anhydrous Na.sub.2 SO.sub.4 and evaporated to dryness. The residue
obtained was purified by flash column chromatography and eluted
with 5% MeOH in CH.sub.2 Cl.sub.2 to get
5'-O-tert-butyldiphenylsilyl-2'-O-[N,N-dimethylaminooxyethyl]-5-methylurid
ine as a white foam (14.6 g, 80%).
2'-O-(dimethylaminooxyethyl)-5-methyluridine
Triethylamine trihydrofluoride (3.91 mL, 24.0 mmol) was dissolved
in dry THF and triethylamine (1.67 mL, 12 mmol, dry, kept over
KOH). This mixture of triethylamine-2HF was then added to
5'-O-tert-butyldiphenylsilyl-2'-O-[N,N-dimethylaminooxyethyl]-5-methylurid
ine (1.40 g, 2.4 mmol) and stirred at room temperature for 24 hrs.
Reaction was monitored by TLC (5% MeOH in CH.sub.2 Cl.sub.2).
Solvent was removed under vacuum and the residue placed on a flash
column and eluted with 10% MeOH in CH.sub.2 Cl.sub.2 to get
2'-O-(dimethylaminooxyethyl)-5-methyluridine (766 mg, 92.5%).
5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine
2'-O-(dimethylaminooxyethyl)-5-methyluridine (750 mg, 2.17 mmol)
was dried over P.sub.2 O.sub.5 under high vacuum overnight at
40.degree. C. It was then co-evaporated with anhydrous pyridine (20
mL). The residue obtained was dissolved in pyridine (11 mL) under
argon atmosphere. 4-dimethylaminopyridine (26.5 mg, 2.60 mmol),
4,4'-dimethoxytrityl chloride (880 mg, 2.60 mmol) was added to the
mixture and the reaction mixture was stirred at room temperature
until all of the starting material disappeared. Pyridine was
removed under vacuum and the residue chromatographed and eluted
with 10% MeOH in CH.sub.2 Cl.sub.2 (containing a few drops of
pyridine) to get
5'-O-DMT-2'-O-(dimethylamino-oxyethyl)-5-methyluridine (1.13 g,
80%).
5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-[(2-cyanoeth
yl)-N,N-diisopropylphosphoramidite]
5'-O-DMT-2'-O-(dimethylaminooxyethyl)-5-methyluridine (1.08 g, 1.67
mmol) was co-evaporated with toluene (20 mL). To the residue
N,N-diisopropylamine tetrazonide (0.29 g, 1.67 mmol) was added and
dried over P.sub.2 O.sub.5 under high vacuum overnight at
40.degree. C. Then the reaction mixture was dissolved in anhydrous
acetonitrile (8.4 mL) and 2-cyanoethyl-N,N,N.sup.1,N.sup.1
-tetraisopropylphosphoramidite (2.12 mL, 6.08 mmol) was added. The
reaction mixture was stirred at ambient temperature for 4 hrs under
inert atmosphere. The progress of the reaction was monitored by TLC
(hexane:ethyl acetate 1:1). The solvent was evaporated, then the
residue was dissolved in ethyl acetate (70 mL) and washed with 5%
aqueous NaHCO.sub.3 (40 mL). Ethyl acetate layer was dried over
anhydrous Na.sub.2 SO.sub.4 and concentrated. Residue obtained was
chromatographed (ethyl acetate as eluent) to get
5'-O-DMT-2'-O-(2-N,N-dimethylaminooxyethyl)-5-methyluridine-3'-[(2-cyanoet
hyl)-N,N-diisopropylphosphoramidite] as a foam (1.04 g, 74.9%).
2'-(Aminooxyethoxy) nucleoside amidites
2'-(Aminooxyethoxy) nucleoside amidites [also known in the art as
2'-O-(aminooxyethyl) nucleoside amidites] are prepared as described
in the following paragraphs. Adenosine, cytidine and thymidine
nucleoside amidites are prepared similarly.
N2-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimetho
xytrityl)guanosine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite]
The 2'-O-aminooxyethyl guanosine analog may be obtained by
selective 2'-O-alkylation of diaminopurine riboside. Multigram
quantities of diaminopurine riboside may be purchased from Schering
AG (Berlin) to provide 2'-O-(2-ethylacetyl) diaminopurine riboside
along with a minor amount of the 3'-O-isomer. 2'-O-(2-ethylacetyl)
diaminopurine riboside may be resolved and converted to
2'-O-(2-ethylacetyl)guanosine by treatment with adenosine
deaminase. (McGee, D. P. C., Cook, P. D., Guinosso, C. J., WO
94/02501 A1 940203.) Standard protection procedures should afford
2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimethoxytrityl)guanosine and
2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimet
hoxytrityl)guanosine which may be reduced to provide
2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimet
hoxytrityl)guanosine. As before the hydroxyl group may be displaced
by N-hydroxyphthalimide via a Mitsunobu reaction, and the protected
nucleoside may phosphitylated as usual to yield
2-N-isobutyryl-6-O-diphenylcarbamoyl-2'-O-(2-ethylacetyl)-5'-O-(4,4'-dimet
hoxytrityl)guanosine-3'-[(2-cyanoethyl)-N,N-diisopropylphosphoramidite].
2'-dimethylaminoethoxyethoxy (2'-DMAEOE) Nucleoside Amidites
2'-dimethylaminoethoxyethoxy nucleoside amidites (also known in the
art as 2'-O-dimethylaminoethoxyethyl, i.e., 2'-O--CH.sub.2
--O--CH.sub.2 --N(CH.sub.2).sub.2, or 2'-DMAEOE nucleoside
amidites) are prepared as follows. Other nucleoside amidites are
prepared similarly.
2'-O-[2(2-N,N-dimethylaminoethoxy)ethyl]-5-methyl Uridine
2[2-(Dimethylamino)ethoxy]ethanol (Aldrich, 6.66 g, 50 mmol) is
slowly added to a solution of borane in tetrahydrofuran (1M, 10 mL,
10 mmol) with stirring in a 100 mL bomb. Hydrogen gas evolves as
the solid dissolves. O.sup.2 -, 2'-anhydro-5-methyluridine (1.2 g,
5 mmol), and sodium bicarbonate (2.5 mg) are added and the bomb is
sealed, placed in an oil bath and heated to 155.degree. C. for 26
hours. The bomb is cooled to room temperature and opened. The crude
solution is concentrated and the residue partitioned between water
(200 mL) and hexanes (200 mL). The excess phenol is extracted into
the hexane layer. The aqueous layer is extracted with ethyl acetate
(3.times.200 mL) and the combined organic layers are washed once
with water, dried over anhydrous sodium sulfate and concentrated.
The residue is columned on silica gel using methanol/methylene
chloride 1:20 (which has 2% triethylamine) as the eluent. As the
column fractions are concentrated a colorless solid forms which is
collected to give the title compound as a white solid.
5'-O-dimethoxytrityl-2'-O-[2(2-N,N-dimethylaminoethoxy)
ethyl)]-5-methyl Uridine
To 0.5 g (1.3 mmol) of
2'-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyl uridine in
anhydrous pyridine (8 mL), triethylamine (0.36 mL) and
dimethoxytrityl chloride (DMT-Cl, 0.87 g, 2 eq.) are added and
stirred for 1 hour. The reaction mixture is poured into water (200
mL) and extracted with CH.sub.2 Cl.sub.2 (2.times.200 mL). The
combined CH.sub.2 Cl.sub.2 layers are washed with saturated
NaHCO.sub.3 solution, followed by saturated NaCl solution and dried
over anhydrous sodium sulfate. Evaporation of the solvent followed
by silica gel chromatography using MeOH:CH.sub.2 Cl.sub.2 :Et.sub.3
N (20:1, v/v, with 1% triethylamine) gives the title compound.
5'-O-Dimethoxytrityl-2'-O-[2(2-N,N-dimethylaminoethoxy)-ethyl)]-5-methyl
uridine-3'-O-(cyanoethyl-N,N-diisopropyl)phosphoramidite
Diisopropylaminotetrazolide (0.6 g) and
2-cyanoethoxy-N,N-diisopropyl phosphoramidite (1.1 mL, 2 eq.) are
added to a solution of
5'-O-dimethoxytrityl-2'-O-[2(2-N,N-dimethylaminoethoxy)ethyl)]-5-methyluri
dine (2.17 g, 3 mmol) dissolved in CH.sub.2 Cl.sub.2 (20 mL) under
an atmosphere of argon. The reaction mixture is stirred overnight
and the solvent evaporated. The resulting residue is purified by
silica gel flash column chromatography with ethyl acetate as the
eluent to give the title compound.
Example 2
Oligonucleotide Synthesis
Unsubstituted and substituted phosphodiester (P=O) oligonucleotides
are synthesized on an automated DNA synthesizer (Applied Biosystems
model 380B) using standard phosphoramidite chemistry with oxidation
by iodine.
Phosphorothioates (P=S) are synthesized as for the phosphodiester
oligonucleotides except the standard oxidation bottle was replaced
by 0.2M solution of 3H-1,2-benzodithiole-3-one 1,1-dioxide in
acetonitrile for the stepwise thiation of the phosphite linkages.
The thiation wait step was increased to 68 sec and was followed by
the capping step. After cleavage from the CPG column and deblocking
in concentrated ammonium hydroxide at 55.degree. C. (18 h), the
oligonucleotides were purified by precipitating twice with 2.5
volumes of ethanol from a 0.5M NaCl solution.
Phosphinate oligonucleotides are prepared as described in U.S. Pat.
No. 5,508,270, herein incorporated by reference.
Alkyl phosphonate oligonucleotides are prepared as described in
U.S. Pat. No. 4,469,863, herein incorporated by reference.
3'-Deoxy-3'-methylene phosphonate oligonucleotides are prepared as
described in U.S. Pat. Nos. 5,610,289 or 5,625,050, herein
incorporated by reference.
Phosphoramidite oligonucleotides are prepared as described in U.S.
Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878, herein incorporated
by reference.
Alkylphosphonothioate oligonucleotides are prepared as described in
published PCT applications PCT/US94/00902 and PCT/US93/06976
(published as WO 94/17093 and WO 94/02499, respectively), herein
incorporated by reference.
3'-Deoxy-3'-amino phosphoramidate oligonucleotides are prepared as
described in U.S. Pat. No. 5,476,925, herein incorporated by
reference.
Phosphotriester oligonucleotides are prepared as described in U.S.
Pat. No. 5,023,243, herein incorporated by reference.
Borano phosphate oligonucleotides are prepared as described in U.S.
Pat. Nos. 5,130,302 and 5,177,198, both herein incorporated by
reference.
Example 3
Oligonucleoside Synthesis
Methylenemethylimino linked oligonucleosides, also identified as
MMI linked oligonucleosides, methylenedimethylhydrazo linked
oligonucleosides, also identified as MDH linked oligonucleosides,
and methylenecarbonylamino linked oligonucleosides, also identified
as amide-3 linked oligonucleosides, and methyleneaminocarbonyl
linked oligonucleosides, also identified as amide-4 linked
oligonucleosides, as well as mixed backbone compounds having, for
instance, alternating MMI and P=O or P=S linkages are prepared as
described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677,
5,602,240 and 5,610,289, all of which are herein incorporated by
reference.
Formacetal and thioformacetal linked oligonucleosides are prepared
as described in U.S. Pat. Nos. 5,264,562 and 5,264,564, herein
incorporated by reference.
Ethylene oxide linked oligonucleosides are prepared as described in
U.S. Pat. No. 5,223,618, herein incorporated by reference.
Example 4
PNA Synthesis
Peptide nucleic acids (PNAs) are prepared in accordance with any of
the various procedures referred to in Peptide Nucleic Acids (PNA):
Synthesis, Properties and Potential Applications, Bioorganic &
Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared in
accordance with U.S. Pat. Nos. 5,539,082, 5,700,922, and 5,719,262,
herein incorporated by reference.
Example 5
Synthesis of Chimeric Oligonucleotides
Chimeric oligonucleotides, oligonucleosides or mixed
oligonucleotides/oligonucleosides of the invention can be of
several different types. These include a first type wherein the
"gap" segment of linked nucleosides is positioned between 5' and 3'
"wing" segments of linked nucleosides and a second "open end" type
wherein the "gap" segment is located at either the 3' or the 5'
terminus of the oligomeric compound. Oligonucleotides of the first
type are also known in the art as "gapmers" or gapped
oligonucleotides. Oligonucleotides of the second type are also
known in the art as "hemimers" or "wingmers".
[2'-O-Me]--[2'-deoxy]--[2'-O-Me] Chimeric Phosphorothioate
Oligonucleotides
Chimeric oligonucleotides having 2'-O-alkyl phosphorothioate and
2'-deoxy phosphorothioate oligonucleotide segments are synthesized
using an Applied Biosystems automated DNA synthesizer Model 380B,
as above. Oligonucleotides are synthesized using the automated
synthesizer and 2'-deoxy-5'-dimethoxytrityl-3'-O-phosphoramidite
for the DNA portion and
5'-dimethoxytrityl-2'-O-methyl-3'-O-phosphoramidite for 5' and 3'
wings. The standard synthesis cycle is modified by increasing the
wait step after the delivery of tetrazole and base to 600 s
repeated four times for RNA and twice for 2'-O-methyl. The fully
protected oligonucleotide is cleaved from the support and the
phosphate group is deprotected in 3:1 ammonia/ethanol at room
temperature overnight then lyophilized to dryness. Treatment in
methanolic ammonia for 24 hrs at room temperature is then done to
deprotect all bases and sample was again lyophilized to dryness.
The pellet is resuspended in 1M TBAF in THF for 24 hrs at room
temperature to deprotect the 2' positions. The reaction is then
quenched with 1M TEAA and the sample is then reduced to 1/2 volume
by rotovac before being desalted on a G25 size exclusion column.
The oligo recovered is then analyzed spectrophotometrically for
yield and for purity by capillary electrophoresis and by mass
spectrometry.
[2'-O-(2-Methoxyethyl)]--[2'-deoxy]--[2'-O-(Methoxyethyl)] Chimeric
Phosphorothioate Oligonucleotides
[2'-O-(2-methoxyethyl)]--[2'-deoxy]--[-2'-O-(methoxyethyl)]
chimeric phosphorothioate oligonucleotides were prepared as per the
procedure above for the 2'-O-methyl chimeric oligonucleotide, with
the substitution of 2'-O-methoxyethyl) amidites for the 2'-O-methyl
amidites.
[2'-O-(2-Methoxyethyl)Phosphodiester]--[2'-deoxy
Phosphorothioate]--[2'-O-(2-Methoxyethyl) Phosphodiester] Chimeric
Oligonucleotides
[2'-O-(2-methoxyethyl phosphodiester]--[2'-deoxy
phosphorothioate]--[2'-O-(methoxyethyl) phosphodiester] chimeric
oligonucleotides are prepared as per the above procedure for the
2'-O-methyl chimeric oligonucleotide with the substitution of
2'-O-(methoxyethyl) amidites for the 2'-O-methyl amidites,
oxidization with iodine to generate the phosphodiester
internucleotide linkages within the wing portions of the chimeric
structures and sulfurization utilizing 3,H-1,2 benzodithiole-3-one
1,1 dioxide (Beaucage Reagent) to generate the phosphorothioate
internucleotide linkages for the center gap.
Other chimeric oligonucleotides, chimeric oligonucleosides and
mixed chimeric oligonucleotides/oligonucleosides are synthesized
according to U.S. Pat. No. 5,623,065, herein incorporated by
reference.
Example 6
Oligonucleotide Isolation
After cleavage from the controlled pore glass column (Applied
Biosystems) and deblocking in concentrated ammonium hydroxide at
55.degree. C. for 18 hours, the oligonucleotides or
oligonucleosides are purified by precipitation twice out of 0.5M
NaCl with 2.5 volumes ethanol. Synthesized oligonucleotides were
analyzed by polyacrylamide gel electrophoresis on denaturing gels
and judged to be at least 85% full length material. The relative
amounts of phosphorothioate and phosphodiester linkages obtained in
synthesis were periodically checked by .sup.31 P nuclear magnetic
resonance spectroscopy, and for some studies oligonucleotides were
purified by HPLC, as described by Chiang et al., J. Biol. Chem.
1991, 266, 18162-18171. Results obtained with HPLC-purified
material were similar to those obtained with non-HPLC purified
material.
Example 7
Oligonucleotide Synthesis--96 Well Plate Format
Oligonucleotides were synthesized via solid phase P(III)
phosphoramidite chemistry on an automated synthesizer capable of
assembling 96 sequences simultaneously in a standard 96 well
format. Phosphodiester internucleotide linkages were afforded by
oxidation with aqueous iodine. Phosphorothioate internucleotide
linkages were generated by sulfurization utilizing 3,H-1,2
benzodithiole-3-one 1,1 dioxide (Beaucage Reagent) in anhydrous
acetonitrile. Standard base-protected beta-cyanoethyldiisopropyl
phosphoramidites were purchased from commercial vendors (e.g.
PE-Applied Biosystems, Foster City, Calif., or Pharmacia,
Piscataway, N.J.). Non-standard nucleosides are synthesized as per
known literature or patented methods. They are utilized as base
protected beta-cyanoethyldiisopropyl phosphoramidites.
Oligonucleotides were cleaved from support and deprotected with
concentrated NH.sub.4 OH at elevated temperature (55-60.degree. C.)
for 12-16 hours and the released product then dried in vacuo. The
dried product was then re-suspended in sterile water to afford a
master plate from which all analytical and test plate samples are
then diluted utilizing robotic pipettors.
Example 8
Oligonucleotide Analysis--96 Well Plate Format
The concentration of oligonucleotide in each well was assessed by
dilution of samples and UV absorption spectroscopy. The full-length
integrity of the individual products was evaluated by capillary
electrophoresis (CE) in either the 96 well format (Beckman
P/ACE.TM. MDQ) or, for individually prepared samples, on a
commercial CE apparatus (e.g., Beckman P/ACE.TM. 5000, ABI 270).
Base and backbone composition was confirmed by mass analysis of the
compounds utilizing electrospray-mass spectroscopy. All assay test
plates were diluted from the master plate using single and
multi-channel robotic pipettors. Plates were judged to be
acceptable if at least 85% of the compounds on the plate were at
least 85% full length.
Example 9
Cell Culture and Oligonucleotide Treatment
The effect of antisense compounds on target nucleic acid expression
can be tested in any of a variety of cell types provided that the
target nucleic acid is present at measurable levels. This can be
routinely determined using, for example, PCR or Northern blot
analysis. The following four cell types are provided for
illustrative purposes, but other cell types can be routinely
used.
T-24 cells
The transitional cell bladder carcinoma cell line T-24 was obtained
from the American Type Culture Collection (ATCC) (Manassas, VA).
T-24 cells were routinely cultured in complete McCoy's 5A basal
media (Gibco/Life Technologies, Gaithersburg, Md.) supplemented
with 10% fetal calf serum (Gibco/Life Technologies, Gaithersburg,
Md.), penicillin 100 units per mL, and streptomycin 100 micrograms
per mL (Gibco/Life Technologies, Gaithersburg, Md.). Cells were
routinely passaged by trypsinization and dilution when they reached
90% confluence. Cells were seeded into 96-well plates
(Falcon-Primaria #3872) at a density of 7000 cells/well for use in
RT-PCR analysis.
For Northern blotting or other analysis, cells may be seeded onto
100 mm or other standard tissue culture plates and treated
similarly, using appropriate volumes of medium and
oligonucleotide.
A549 cells
The human lung carcinoma cell line A549 was obtained from the
American Type Culture Collection (ATCC) (Manassas, Va.). A549 cells
were routinely cultured in DMEM basal media (Gibco/Life
Technologies, Gaithersburg, Md.) supplemented with 10% fetal calf
serum (Gibco/Life Technologies, Gaithersburg, Md.), penicillin 100
units per mL, and streptomycin 100 micrograms per mL (Gibco/Life
Technologies, Gaithersburg, Md.). Cells were routinely passaged by
trypsinization and dilution when they reached 90% confluence.
NHDF cells
Human neonatal dermal fibroblast (NHDF) were obtained from the
Clonetics Corporation (Walkersville, Md.). NHDFs were routinely
maintained in Fibroblast Growth Medium (Clonetics Corporation,
Walkersville, Md.) supplemented as recommended by the supplier.
Cells were maintained for up to 10 passages as recommended by the
supplier.
HEK cells
Human embryonic keratinocytes (HEK) were obtained from the
Clonetics Corporation (Walkersville, Md.). HEKs were routinely
maintained in Keratinocyte Growth Medium (Clonetics Corporation,
Walkersville, Md.) formulated as recommended by the supplier. Cells
were routinely maintained for up to 10 passages as recommended by
the supplier.
Treatment with antisense compounds
When cells reached 80% confluency, they were treated with
oligonucleotide. For cells grown in 96-well plates, wells were
washed once with 200 .mu.L OPTI-MEM.TM.-1 reduced-serum medium
(Gibco BRL) and then treated with 130 .mu.L of OPTI-MEM.TM.-1
containing 3.75 .mu.g/mL LIPOFECTIN.TM. (Gibco BRL) and the desired
oligonucleotide at a final concentration of 150 nM. After 4 hours
of treatment, the medium was replaced with fresh medium. Cells were
harvested 16 hours after oligonucleotide treatment.
Example 10
Analysis of Oligonucleotide Inhibition of Integrin beta 3
Expression
Antisense modulation of integrin beta 3 expression can be assayed
in a variety of ways known in the art. For example, integrin beta 3
mRNA levels can be quantitated by, e.g., Northern blot analysis,
competitive polymerase chain reaction (PCR), or real-time PCR
(RT-PCR). Real-time quantitative PCR is presently preferred. RNA
analysis can be performed on total cellular RNA or poly(A)+ mRNA.
Methods of RNA isolation are taught in, for example, Ausubel, F. M.
et al., Current Protocols in Molecular Biology, Volume 1, pp.
4.1.1-4.2.9 and 4.5.1-4.5.3, John Wiley & Sons, Inc., 1993.
Northern blot analysis is routine in the art and is taught in, for
example, Ausubel, F. M. et al., Current Protocols in Molecular
Biology, Volume 1, pp. 4.2.1-4.2.9, John Wiley & Sons, Inc.,
1996. Real-time quantitative (PCR) can be conveniently accomplished
using the commercially available ABI PRISM.TM. 7700 Sequence
Detection System, available from PE-Applied Biosystems, Foster
City, Calif. and used according to manufacturer's instructions.
Other methods of PCR are also known in the art.
integrin beta 3 protein levels can be quantitated in a variety of
ways well known in the art, such as immunoprecipitation, Western
blot analysis (immunoblotting), ELISA or fluorescence-activated
cell sorting (FACS). Antibodies directed to integrin beta 3 can be
identified and obtained from a variety of sources, such as the MSRS
catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or
can be prepared via conventional antibody generation methods.
Methods for preparation of polyclonal antisera are taught in, for
example, Ausubel, F. M. et al., Current Protocols in Molecular
Biology, Volume 2, pp. 11.12.1-11.12.9, John Wiley & Sons,
Inc., 1997. Preparation of monoclonal antibodies is taught in, for
example, Ausubel, F. M. et al., Current Protocols in Molecular
Biology, Volume 2, pp. 11.4.1-11.11.5, John Wiley & Sons, Inc.,
1997.
Immunoprecipitation methods are standard in the art and can be
found at, for example, Ausubel, F. M. et al., Current Protocols in
Molecular Biology, Volume 2, pp. 10.16.1-10.16.11, John Wiley &
Sons, Inc., 1998. Western blot (immunoblot) analysis is standard in
the art and can be found at, for example, Ausubel, F. M. et al.,
Current Protocols in Molecular Biology, Volume 2, pp.
10.8.1-10.8.21, John Wiley & Sons, Inc., 1997. Enzyme-linked
immunosorbent assays (ELISA) are standard in the art and can be
found at, for example, Ausubel, F. M. et al., Current Protocols in
Molecular Biology, Volume 2, pp. 11.2.1-11.2.22, John Wiley &
Sons, Inc., 1991.
Example 11
Poly(A)+ mRNA Isolation
Poly(A)+ mRNA was isolated according to Miura et al., Clin. Chem.,
1996, 42, 1758-1764. Other methods for poly(A)+ mRNA isolation are
taught in, for example, Ausubel, F. M. et al., Current Protocols in
Molecular Biology, Volume 1, pp. 4.5.1-4.5.3, John Wiley &
Sons, Inc., 1993. Briefly, for cells grown on 96-well plates,
growth medium was removed from the cells and each well was washed
with 200 .mu.L cold PBS. 60 .mu.L lysis buffer (10 mM Tris-HCl, pH
7.6, l mM EDTA, 0.5M NaCl, 0.5% NP-40, 20 mM vanadyl-ribonucleoside
complex) was added to each well, the plate was gently agitated and
then incubated at room temperature for five minutes. 55 .mu.L of
lysate was transferred to Oligo d(T) coated 96-well plates (AGCT
Inc., Irvine, Calif.). Plates were incubated for 60 minutes at room
temperature, washed 3 times with 200 .mu.L of wash buffer (10 mM
Tris-HCl pH 7.6, 1 mM EDTA, 0.3M NaCl). After the final wash, the
plate was blotted on paper towels to remove excess wash buffer and
then air-dried for 5 minutes. 60 .mu.L of elution buffer (5 mM
Tris-HCl pH 7.6), preheated to 70.degree. C. was added to each
well, the plate was incubated on a 90.degree. C. hot plate for 5
minutes, and the eluate was then transferred to a fresh 96-well
plate.
Cells grown on 100 mm or other standard plates may be treated
similarly, using appropriate volumes of all solutions.
Example 12
Total RNA Isolation
Total mRNA was isolated using an RNEASY 96.TM. kit and buffers
purchased from Qiagen Inc. (Valencia, Calif.) following the
manufacturer's recommended procedures. Briefly, for cells grown on
96-well plates, growth medium was removed from the cells and each
well was washed with 200 .mu.L cold PBS. 100 .mu.L Buffer RLT was
added to each well and the plate vigorously agitated for 20
seconds. 100 .mu.L of 70% ethanol was then added to each well and
the contents mixed by pipetting three times up and down. The
samples were then transferred to the RNEASY 96.TM. well plate
attached to a QIAVAC.TM. manifold fitted with a waste collection
tray and attached to a vacuum source. Vacuum was applied for 15
seconds. 1 mL of Buffer RW1 was added to each well of the RNEASY
96.TM. plate and the vacuum again applied for 15 seconds. 1 mL of
Buffer RPE was then added to each well of the RNEASY 96.TM. plate
and the vacuum applied for a period of 15 seconds. The Buffer RPE
wash was then repeated and the vacuum was applied for an additional
10 minutes. The plate was then removed from the QIAVAC.TM. manifold
and blotted dry on paper towels. The plate was then re-attached to
the QIAVAC.TM. manifold fitted with a collection tube rack
containing 1.2 mL collection tubes. RNA was then eluted by
pipetting 60 .mu.L water into each well, incubating 1 minute, and
then applying the vacuum for 30 seconds. The elution step was
repeated with an additional 60 .mu.L water.
The repetitive pipetting and elution steps may be automated using a
QIAGEN Bio-Robot 9604 (Qiagen, Inc., Valencia, Calif.). Essentially
after lysing of the cells on the culture plate, the plate is
transferred to the robot deck where the pipetting, DNase treatment
and elution steps are carried out.
Example 13
Real-time Quantitative PCR Analysis of Integrin beta 3 mRNA
Levels
Quantitation of integrin beta 3 mRNA levels was determined by
real-time quantitative PCR using the ABI PRISM.TM. 7700 Sequence
Detection System (PE-Applied Biosystems, Foster City, Calif.)
according to manufacturer's instructions. This is a closed-tube,
non-gel-based, fluorescence detection system which allows
high-throughput quantitation of polymerase chain reaction (PCR)
products in real-time. As opposed to standard PCR, in which
amplification products are quantitated after the PCR is completed,
products in real-time quantitative PCR are quantitated as they
accumulate. This is accomplished by including in the PCR reaction
an oligonucleotide probe that anneals specifically between the
forward and reverse PCR primers, and contains two fluorescent dyes.
A reporter dye (e.g., JOE or FAM, obtained from either Operon
Technologies Inc., Alameda, Calif. or PE-Applied Biosystems, Foster
City, Calif.) is attached to the 5' end of the probe and a quencher
dye (e.g., TAMRA, obtained from either Operon Technologies Inc.,
Alameda, Calif. or PE-Applied Biosystems, Foster City, Calif.) is
attached to the 3' end of the probe. When the probe and dyes are
intact, reporter dye emission is quenched by the proximity of the
3' quencher dye. During amplification, annealing of the probe to
the target sequence creates a substrate that can be cleaved by the
5'-exonuclease activity of Taq polymerase. During the extension
phase of the PCR amplification cycle, cleavage of the probe by Taq
polymerase releases the reporter dye from the remainder of the
probe (and hence from the quencher moiety) and a sequence-specific
fluorescent signal is generated. With each cycle, additional
reporter dye molecules are cleaved from their respective probes,
and the fluorescence intensity is monitored at regular intervals by
laser optics built into the ABI PRISM.TM. 7700 Sequence Detection
System. In each assay, a series of parallel reactions containing
serial dilutions of mRNA from untreated control samples generates a
standard curve that is used to quantitate the percent inhibition
after antisense oligonucleotide treatment of test samples.
PCR reagents were obtained from PE-Applied Biosystems, Foster City,
Calif.. RT-PCR reactions were carried out by adding 25 .mu.L PCR
cocktail (1.times. TAQMAN.TM. buffer A, 5.5 mM MgCl.sub.2, 300
.mu.M each of DATP, dCTP and dGTP, 600 .mu.M of dUTP, 100 nM each
of forward primer, reverse primer, and probe, 20 Units RNAse
inhibitor, 1.25 Units AMPLITAQ GOLD.TM., and 12.5 Units MuLV
reverse transcriptase) to 96 well plates containing 25 .mu.L
poly(A) mRNA solution. The RT reaction was carried out by
incubation for 30 minutes at 48.degree. C. Following a 10 minute
incubation at 95.degree. C. to activate the AMPLITAQ GOLD.TM., 40
cycles of a two-step PCR protocol were carried out: 95.degree. C.
for 15 seconds (denaturation) followed by 60.degree. C. for 1.5
minutes (annealing/extension). integrin beta 3 probes and primers
were designed to hybridize to the human integrin beta 3 sequence,
using published sequence information (GenBank accession number
J02703, incorporated herein as SEQ ID NO:1).
For integrin beta 3 the PCR primers were: forward primer:
TTTACCACTGATGCCAAGACTCA (SEQ ID NO: 2) reverse primer:
CCGTCATTAGGCTGGACAATG (SEQ ID NO: 3) and the PCR probe was:
FAM-ATAGCATTGGACGGAAGGCTGGCAG-TAMRA (SEQ ID NO: 4) where FAM
(PE-Applied Biosystems, Foster City, Calif.) is the fluorescent
reporter dye) and TAMRA (PE-Applied Biosystems, Foster City,
Calif.) is the quencher dye.
For GAPDH the PCR primers were: forward primer: GAAGGTGAAGGTCGGAGTC
(SEQ ID NO: 5) reverse primer: GAAGATGGTGATGGGATTTC (SEQ ID NO: 6)
and the PCR probe was: 5' JOE-CAAGCTTCCCGTTCTCAGCC- TAMRA 3' (SEQ
ID NO: 7) where JOE (PE-Applied Biosystems, Foster City, Calif.) is
the fluorescent reporter dye) and TAMRA (PE-Applied Biosystems,
Foster City, Calif.) is the quencher dye.
Example 14
Northern Blot Analysis of Integrin beta 3 mRNA Levels
Eighteen hours after antisense treatment, cell monolayers were
washed twice with cold PBS and lysed in 1 mL RNAZOL.TM. (TEL-TEST
"B" Inc., Friendswood, Tex.). Total RNA was prepared following
manufacturer's recommended protocols. Twenty micrograms of total
RNA was fractionated by electrophoresis through 1.2% agarose gels
containing 1.1% formaldehyde using a MOPS buffer system (AMRESCO,
Inc. Solon, Ohio). RNA was transferred from the gel to
HYBOND.TM.-N+ nylon membranes (Amersham Pharmacia Biotech,
Piscataway, N.J.) by overnight capillary transfer using a
Northern/Southern Transfer buffer system (TEL-TEST "B" Inc.,
Friendswood, Tex.). RNA transfer was confirmed by UV visualization.
Membranes were fixed by UV cross-linking using a STRATALINKER.TM.
UV Crosslinker 2400 (Stratagene, Inc, La Jolla, Calif.).
Membranes were probed using QUICKHYB.TM. hybridization solution
(Stratagene, La Jolla, Calif.) using manufacturer's recommendations
for stringent conditions with a integrin beta 3 specific probe
prepared by PCR using the forward primer TTTACCACTGATGCCAAGACTCA
(SEQ ID NO: 2) and the reverse primer CCGTCATTAGGCTGGACAATG (SEQ ID
NO: 3). To normalize for variations in loading and transfer
efficiency membranes were stripped and probed for
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) RNA (Clontech,
Palo Alto, Calif.). Hybridized membranes were visualized and
quantitated using a PHOSPHORIMAGER.TM. and IMAGEQUANT.TM. Software
V3.3 (Molecular Dynamics, Sunnyvale, Calif.). Data was normalized
to GAPDH levels in untreated controls.
Example 15
Antisense Inhibition of Integrin beta 3 Expression-phosphorothioate
Oligodeoxynucleotides
In accordance with the present invention, a series of
oligonucleotides were designed to target different regions of the
human integrin beta 3 RNA, using published sequences (GenBank
accession number J02703, incorporated herein as SEQ ID NO: 1). The
oligonucleotides are shown in Table 1. Target sites are indicated
by nucleotide numbers, as given in the sequence source reference
(Genbank accession no. J02703), to which the oligonucleotide binds.
All compounds in Table 1 are oligodeoxynucleotides with
phosphorothioate backbones (internucleoside linkages) throughout.
The compounds were analyzed for effect on integrin beta 3 mRNA
levels by quantitative real-time PCR as described in other examples
herein. Data are averages from two experiments. If present, "N.D."
indicates "no data".
TABLE 1 ______________________________________ Inhibition of
integrin beta 3 mRNA levels by phosphorothioate
oligodeoxynucleotides ISIS TARGET % Inhi- SEQ ID # REGION SITE
SEQUENCE bition NO. ______________________________________ 25177
Start 7 gcatctcgtccgcctccc 56 8 Codon 25178 Coding 50
gccagcacagtcacccag 0 9 25179 Coding 258 actcactgggaactcgat 34 10
25180 Coding 389 tggatggagaaattcttc 36 11 25181 Coding 480
gttctggatgctccacag 45 12 25182 Coding 545 gccccgaagccaatccgc 0 13
25183 Coding 689 aagcgggtcacctggtca 0 14 25184 Coding 802
cattcctccagccaatct 40 15 25185 Coding 863 cttccgtccaatgctata 4 16
25186 Coding 916 tgtcactaccaacatgac 68 17 25187 Coding 977
agcttctcagtcatcagc 72 18 25188 Coding 1033 agagattgactacatttt 7 19
25189 Coding 1144 ctttagaacggattttcc 0 20 25190 Coding 1182
agacaactcttcagggag 38 21 25191 Coding 1267 tcaccgtgtctccaatct 47 22
25192 Coding 1339 ccacgggctttatggtaa 37 23 25193 Coding 1406
tcagcttgggcctggcag 40 24 25194 Coding 1513 agtcctcctctgagcact 45 25
25195 Coding 1600 cacattgaccacagaggc 26 26 25196 Coding 1660
agaagtcgtcacactcgc 28 27 25197 Coding 1798 gcccattgctggacatgc 0 28
25198 Coding 1903 aggcatctgggcaggtgg 10 29 25199 Coding 1943
tcaaacttcttacactcc 46 30 25200 Coding 2036 gcatccttgccagtgtcc 53 31
25201 Coding 2075 acgacacagtcatcctca 0 32 25202 Coding 2126
accacatacaggatggac 0 33 25203 Coding 2240 gtgatgaggagtttccag 2 34
25204 Coding 2302 tgtcccattttgctctgg 47 35 25205 Coding 2342
aaggtagacgtggcctct 29 36 25206 Stop 2384 atgactgcttatcattaa 0 37
Codon 25207 3' UTR 2424 gactcctgcaatcctggc 47 38 25208 3' UTR 2510
acatactgacattctccc 0 39 25209 3' UTR 2564 acacacacataaacacac 0 40
25210 3' UTR 2679 tcaccctcaagctaagca 0 41 25211 3' UTR 2719
tctcactgaggtaatgaa 34 42 25212 3' UTR 2859 agctgctgagagtcaggc 78 43
25213 3' UTR 2934 tccttcggcctgagggag 43 44 25214 3' UTR 2976
aaaggcacaggcagctct 0 45 25215 3' UTR 3108 agatgctggtacatcccc 60 46
25216 3' UTR 3147 caataaggcaaatctgaa 23 47
______________________________________
As shown in Table 1, SEQ ID NOs 8, 10, 11, 12, 15, 17, 18, 21, 22,
23, 24, 25, 26, 27, 30, 31, 35, 36, 38, 42, 43, 44, 46 and 47
demonstrated at least 20% inhibition of in beta 3 expression in
this assay and are therefore preferred.
Example 16
Antisense Inhibition of Integrin beta 3 Expression-phosphorothioate
2'-MOE Gapmer Oligonucleotides
In accordance with the present invention, a second series of
oligonucleotides targeted to human integrin beta 3 were
synthesized. The oligonucleotide sequences are shown in Table 2.
Target sites are indicated by nucleotide numbers, as given in the
sequence source reference (Genbank accession no. J02703), to which
the oligonucleotide binds.
All compounds in Table 2 are chimeric oligonucleotides ("gapmers")
18 nucleotides in length, composed of a central "gap" region
consisting of ten 2'-deoxynucleotides, which is flanked on both
sides (5' and 3' directions) by four-nucleotide "wings". The wings
are composed of 2'-methoxyethyl (2'-MOE)nucleotides. The
internucleoside (backbone) linkages are phosphorothioate (P=S)
throughout the oligonucleotide. Cytidine residues in the 2'-MOE
wings are 5-methylcytidines.
Data were obtained by real-time quantitative PCR as described in
other examples herein and are averaged from two experiments. If
present, "N.D." indicates "no data".
TABLE 2 ______________________________________ Inhibition of
integrin beta 3 mRNA levels by chimeric phosphorothioate
oligonucleotides having 2'-MOE wings and a deoxy gap ISIS TARGET %
Inhi- SEQ ID # REGION SITE SEQUENCE bition NO.
______________________________________ 25217 Start 7
gcatctcgtccgcctccc 17 8 Codon 25218 Coding 50 gccagcacagtcacccag 49
9 25219 Coding 258 actcactgggaactcgat 0 10 25220 Coding 389
tggatggagaaattcttc 33 11 25221 Coding 480 gttctggatgctccacag 58 12
25222 Coding 545 gccccgaagccaatccgc 35 13 25223 Coding 689
aagcgggtcacctggtca 0 14 25224 Coding 802 cattcctccagccaatct 0 15
25225 Coding 863 cttccgtccaatgctata 0 16 25226 Coding 916
tgtcactaccaacatgac 3 17 25227 Coding 977 agcttctcagtcatcagc 0 18
25228 Coding 1033 agagattgactacatttt 0 19 25229 Coding 1144
ctttagaacggattttcc 30 20 25230 Coding 1182 agacaactcttcagggag 5 21
25231 Coding 1267 tcaccgtgtctccaatct 0 22 25232 Coding 1339
ccacgggctttatggtaa 28 23 25233 Coding 1406 tcagcttgggcctggcag 0 24
25234 Coding 1513 agtcctcctctgagcact 35 25 25235 Coding 1600
cacattgaccacagaggc 0 26 25236 Coding 1660 agaagtcgtcacactcgc 63 27
25237 Coding 1798 gcccattgctggacatgc 80 28 25238 Coding 1903
aggcatctgggcaggtgg 48 29 25239 Coding 1943 tcaaacttcttacactcc 22 30
25240 Coding 2036 gcatccttgccagtgtcc 0 31 25241 Coding 2075
acgacacagtcatcctca 0 32 25242 Coding 2126 accacatacaggatggac 58 33
25243 Coding 2240 gtgatgaggagtttccag 8 34 25244 Coding 2302
tgtcccattttgctctgg 56 35 25245 Coding 2342 aaggtagacgtggcctct 45 36
25246 Stop 2384 atgactgcttatcattaa 11 37 Codon 25247 3' UTR 2424
gactcctgcaatcctggc 8 38 25248 3' UTR 2510 acatactgacattctccc 0 39
25249 3' UTR 2564 acacacacataaacacac 0 40 25250 3' UTR 2679
tcaccctcaagctaagca 52 41 25251 3' UTR 2719 tctcactgaggtaatgaa 19 42
25252 3' UTR 2859 agctgctgagagtcaggc 0 43 25253 3' UTR 2934
tccttcggcctgagggag 23 44 25254 3' UTR 2976 aaaggcacaggcagctct 0 45
25255 3' UTR 3108 agatgctggtacatcccc 0 46 25256 3' UTR 3147
caataaggcaaatctgaa 0 47 ______________________________________
As shown in Table 2, SEQ ID NOs 9, 11, 12, 13, 20, 23, 25, 27, 28,
29, 30, 33, 35, 36, 41 and 44 demonstrated at least 20% inhibition
of integrin beta 3 expression in this experiment and are therefore
preferred.
Example 17
Western Blot Analysis of Integrin beta 3 Protein Levels
Western blot analysis (immunoblot analysis) is carried out using
standard methods. Cells are harvested 16-20 h after oligonucleotide
treatment, washed once with PBS, suspended in Laemmli buffer (100
ul/well), boiled for 5 minutes and loaded on a 16% SDS-PAGE gel.
Gels are run for 1.5 hours at 150 V, and transferred to membrane
for western blotting. Appropriate primary antibody directed to
integrin beta 3 is used, with a radiolabelled or fluorescently
labeled secondary antibody directed against the primary antibody
species. Bands are visualized using a PHOSPHORIMAGER.TM. (Molecular
Dynamics, Sunnyvale, Calif.).
__________________________________________________________________________
# SEQUENCE LISTING - - - - <160> NUMBER OF SEQ ID NOS: 47 - -
<210> SEQ ID NO 1 <211> LENGTH: 3170 <212> TYPE:
DNA <213> ORGANISM: Homo sapiens <220> FEATURE:
<221> NAME/KEY: CDS <222> LOCATION: (21)..(2387) - -
<400> SEQUENCE: 1 - - cgccgcggga ggcggacgag atg cga gcg cgg
ccg cgg cc - #c cgg ccg ctc 50 - # Met Arg Ala Arg Pro Arg Pro Arg
- #Pro Leu - # 1 - # 5 - # 10 - - tgg gtg act gtg ctg gcg ctg ggg
gcg ctg gc - #g ggc gtt ggc gta gga 98 Trp Val Thr Val Leu Ala Leu
Gly Ala Leu Al - #a Gly Val Gly Val Gly 15 - # 20 - # 25 - - ggg
ccc aac atc tgt acc acg cga ggt gtg ag - #c tcc tgc cag cag tgc 146
Gly Pro Asn Ile Cys Thr Thr Arg Gly Val Se - #r Ser Cys Gln Gln Cys
30 - # 35 - # 40 - - ctg gct gtg agc ccc atg tgt gcc tgg tgc tc -
#t gat gag gcc ctg cct 194 Leu Ala Val Ser Pro Met Cys Ala Trp Cys
Se - #r Asp Glu Ala Leu Pro 45 - # 50 - # 55 - - ctg ggc tca cct
cgc tgt gac ctg aag gag aa - #t ctg ctg aag gat aac 242 Leu Gly Ser
Pro Arg Cys Asp Leu Lys Glu As - #n Leu Leu Lys Asp Asn 60 - # 65 -
# 70 - - tgt gcc cca gaa tcc atc gag ttc cca gtg ag - #t gag gcc
cga gta cta 290 Cys Ala Pro Glu Ser Ile Glu Phe Pro Val Se - #r Glu
Ala Arg Val Leu 75 - # 80 - # 85 - # 90 - - gag gac agg ccc ctc agc
gac aag ggc tct gg - #a gac agc tcc cag gtc 338 Glu Asp Arg Pro Leu
Ser Asp Lys Gly Ser Gl - #y Asp Ser Ser Gln Val 95 - # 100 - # 105
- - act caa gtc agt ccc cag agg att gca ctc cg - #g ctc cgg cca gat
gat 386 Thr Gln Val Ser Pro Gln Arg Ile Ala Leu Ar - #g Leu Arg Pro
Asp Asp 110 - # 115 - # 120 - - tcg aag aat ttc tcc atc caa gtg cgg
cag gt - #g gag gat tac cct gtg 434 Ser Lys Asn Phe Ser Ile Gln Val
Arg Gln Va - #l Glu Asp Tyr Pro Val 125 - # 130 - # 135 - - gac atc
tac tac ttg atg gac ctg tct tac tc - #c atg aag gat gat ctg 482 Asp
Ile Tyr Tyr Leu Met Asp Leu Ser Tyr Se - #r Met Lys Asp Asp Leu 140
- # 145 - # 150 - - tgg agc atc cag aac ctg ggt acc aag ctg gc - #c
acc cag atg cga aag 530 Trp Ser Ile Gln Asn Leu Gly Thr Lys Leu Al
- #a Thr Gln Met Arg Lys 155 1 - #60 1 - #65 1 - #70 - - ctc acc
agt aac ctg cgg att ggc ttc ggg gc - #a ttt gtg gac aag cct 578 Leu
Thr Ser Asn Leu Arg Ile Gly Phe Gly Al - #a Phe Val Asp Lys Pro 175
- # 180 - # 185 - - gtg tca cca tac atg tat atc tcc cca cca ga - #g
gcc ctc gaa aac ccc 626 Val Ser Pro Tyr Met Tyr Ile Ser Pro Pro Gl
- #u Ala Leu Glu Asn Pro 190 - # 195 - # 200 - - tgc tat gat atg
aag acc acc tgc ttg ccc at - #g ttt ggc tac aaa cac 674 Cys Tyr Asp
Met Lys Thr Thr Cys Leu Pro Me - #t Phe Gly Tyr Lys His 205 - # 210
- # 215 - - gtg ctg acg cta act gac cag gtg acc cgc tt - #c aat gag
gaa gtg aag 722 Val Leu Thr Leu Thr Asp Gln Val Thr Arg Ph - #e Asn
Glu Glu Val Lys 220 - # 225 - # 230 - - aag cag agt gtg tca cgg aac
cga gat gcc cc - #a gag ggt ggc ttt gat 770 Lys Gln Ser Val Ser Arg
Asn Arg Asp Ala Pr - #o Glu Gly Gly Phe Asp 235 2 - #40 2 - #45 2 -
#50 - - gcc atc atg cag gct aca gtc tgt gat gaa aa - #g att ggc tgg
agg aat 818 Ala Ile Met Gln Ala Thr Val Cys Asp Glu Ly - #s Ile Gly
Trp Arg Asn 255 - # 260 - # 265 - - gat gca tcc cac ttg ctg gtg ttt
acc act ga - #t gcc aag act cat ata 866 Asp Ala Ser His Leu Leu Val
Phe Thr Thr As - #p Ala Lys Thr His Ile 270 - # 275 - # 280 - - gca
ttg gac gga agg ctg gca ggc att gtc ca - #g cct aat gac ggg cag 914
Ala Leu Asp Gly Arg Leu Ala Gly Ile Val Gl - #n Pro Asn Asp Gly Gln
285 - # 290 - # 295 - - tgt cat gtt ggt agt gac aat cat tac tct gc
- #c tcc act acc atg gat 962 Cys His Val Gly Ser Asp Asn His Tyr
Ser Al - #a Ser Thr Thr Met Asp 300 - # 305 - # 310 - - tat ccc tct
ttg ggg ctg atg act gag aag ct - #a tcc cag aaa aac atc 1010 Tyr
Pro Ser Leu Gly Leu Met Thr Glu Lys Le - #u Ser Gln Lys Asn Ile 315
3 - #20 3 - #25 3 - #30 - - aat ttg atc ttt gca gtg act gaa aat gta
gt - #c aat ctc tat cag aac 1058 Asn Leu Ile Phe Ala Val Thr Glu
Asn Val Va - #l Asn Leu Tyr Gln Asn 335 - # 340 - # 345 - - tat agt
gag ctc atc cca ggg acc aca gtt gg - #g gtt ctg tcc atg gat 1106
Tyr Ser Glu Leu Ile Pro Gly Thr Thr Val Gl - #y Val Leu Ser Met Asp
350 - # 355 - # 360 - - tcc agc aat gtc ctc cag ctc att gtt gat gc
- #t tat ggg aaa atc cgt 1154 Ser Ser Asn Val Leu Gln Leu Ile Val
Asp Al - #a Tyr Gly Lys Ile Arg 365 - # 370 - # 375 - - tct aaa gtc
gag ctg gaa gtg cgt gac ctc cc - #t gaa gag ttg tct cta 1202 Ser
Lys Val Glu Leu Glu Val Arg Asp Leu Pr - #o Glu Glu Leu Ser Leu 380
- # 385 - # 390 - - tcc ttc aat gcc acc tgc ctc aac aat gag gt - #c
atc cct ggc ctc aag 1250 Ser Phe Asn Ala Thr Cys Leu Asn Asn Glu Va
- #l Ile Pro Gly Leu Lys 395 4 - #00 4 - #05 4 - #10 - - tct tgt
atg gga ctc aag att gga gac acg gt - #g agc ttc agc att gag 1298
Ser Cys Met Gly Leu Lys Ile Gly Asp Thr Va - #l Ser Phe Ser Ile Glu
415 - # 420 - # 425 - - gcc aag gtg cga ggc tgt ccc cag gag aag ga
- #g aag tcc ttt acc ata 1346 Ala Lys Val Arg Gly Cys Pro Gln Glu
Lys Gl - #u Lys Ser Phe Thr Ile 430 - # 435 - # 440 - - aag ccc gtg
ggc ttc aag gac agc ctg atc gt - #c cag gtc acc ttt gat 1394 Lys
Pro Val Gly Phe Lys Asp Ser Leu Ile Va - #l Gln Val Thr Phe Asp 445
- # 450 - # 455 - - tgt gac tgt gcc tgc cag gcc caa gct gaa cc - #t
aat agc cat cgc tgc 1442 Cys Asp Cys Ala Cys Gln Ala Gln Ala Glu Pr
- #o Asn Ser His Arg Cys 460 - # 465 - # 470 - - aac aat ggc aat
ggg acc ttt gag tgt ggg gt - #a tgc cgt tgt ggg cct 1490 Asn Asn
Gly Asn Gly Thr Phe Glu Cys Gly Va - #l Cys Arg Cys Gly Pro 475 4 -
#80 4 - #85 4 - #90 - - ggc tgg ctg gga tcc cag tgt gag tgc tca ga
- #g gag gac tat cgc cct 1538 Gly Trp Leu Gly Ser Gln Cys Glu Cys
Ser Gl - #u Glu Asp Tyr Arg Pro 495 - # 500 - # 505 - - tcc cag cag
gac gag tgc agc ccc cga gag gg - #t cag ccc gtc tgc agc 1586 Ser
Gln Gln Asp Glu Cys Ser Pro Arg Glu Gl - #y Gln Pro Val Cys Ser 510
- # 515 - # 520 - - cag cgg ggc gag tgc ctc tgt ggt caa tgt gt - #c
tgc cac agc agt gac 1634 Gln Arg Gly Glu Cys Leu Cys Gly Gln Cys Va
- #l Cys His Ser Ser Asp 525 - # 530 - # 535 - - ttt ggc aag atc
acg ggc aag tac tgc gag tg - #t gac gac ttc tcc tgt 1682 Phe Gly
Lys Ile Thr Gly Lys Tyr Cys Glu Cy - #s Asp Asp Phe Ser Cys 540 - #
545 - # 550 - - gtc cgc tac aag ggg gag atg tgc tca ggc ca - #t ggc
cag tgc agc tgt 1730 Val Arg Tyr Lys Gly Glu Met Cys Ser Gly Hi -
#s Gly Gln Cys Ser Cys 555 5 - #60 5 - #65 5 - #70 - - ggg gac tgc
ctg tgt gac tcc gac tgg acc gg - #c tac tac tgc aac tgt 1778 Gly
Asp Cys Leu Cys Asp Ser Asp Trp Thr Gl - #y Tyr Tyr Cys Asn Cys 575
- # 580 - # 585 - - acc acg cgt act gac acc tgc atg tcc agc aa - #t
ggg ctg ctg tgc agc 1826 Thr Thr Arg Thr Asp Thr Cys Met Ser Ser As
- #n Gly Leu Leu Cys Ser 590 - # 595 - # 600 - - ggc cgc ggc aag
tgt gaa tgt ggc agc tgt gt - #c tgt atc cag ccg ggc 1874 Gly Arg
Gly Lys Cys Glu Cys Gly Ser Cys Va - #l Cys Ile Gln Pro Gly 605 - #
610 - # 615 - - tcc tat ggg gac acc tgt gag aag tgc ccc ac - #c tgc
cca gat gcc tgc 1922 Ser Tyr Gly Asp Thr Cys Glu Lys Cys Pro Th -
#r Cys Pro Asp Ala Cys 620 - # 625 - # 630 - - acc ttt aag aaa gaa
tgt gtg gag tgt aag aa - #g ttt gac cgg gag ccc 1970 Thr Phe Lys
Lys Glu Cys Val Glu Cys Lys Ly - #s Phe Asp Arg Glu Pro 635 6 - #40
6 - #45 6 - #50 - - tac atg acc gaa aat acc tgc aac cgt tac tg - #c
cgt gac gag att gag 2018 Tyr Met Thr Glu Asn Thr Cys Asn Arg Tyr Cy
- #s Arg Asp Glu Ile Glu 655 - # 660 - # 665 - - tca gtg aaa gag
ctt aag gac act ggc aag ga - #t gca gtg aat tgt acc 2066 Ser Val
Lys Glu Leu Lys Asp Thr Gly Lys As - #p Ala Val Asn Cys Thr 670 - #
675 - # 680 - - tat aag aat gag gat gac tgt gtc gtc aga tt - #c cag
tac tat gaa gat 2114 Tyr Lys Asn Glu Asp Asp Cys Val Val Arg Ph -
#e Gln Tyr Tyr Glu Asp 685 - # 690 - # 695 - - tct agt gga aag tcc
atc ctg tat gtg gta ga - #a gag cca gag tgt ccc 2162 Ser Ser Gly
Lys Ser Ile Leu Tyr Val Val Gl - #u Glu Pro Glu Cys Pro 700 - # 705
- # 710 - - aag ggc cct gac atc ctg gtg gtc ctg ctc tc - #a gtg atg
ggg gcc att 2210 Lys Gly Pro Asp Ile Leu Val Val Leu Leu Se - #r
Val Met Gly Ala Ile 715 7 - #20 7 - #25 7 - #30 - - ctg ctc att ggc
ctt gcc gcc ctg ctc atc tg - #g aaa ctc ctc atc acc 2258 Leu Leu
Ile Gly Leu Ala Ala Leu Leu Ile Tr - #p Lys Leu Leu Ile Thr 735 - #
740 - # 745 - - atc cac gac cga aaa gaa ttc gct aaa ttt ga - #g gaa
gaa cgc gcc aga 2306 Ile His Asp Arg Lys Glu Phe Ala Lys Phe Gl -
#u Glu Glu Arg Ala Arg 750 - # 755 - # 760 - - gca aaa tgg gac aca
gcc aac aac cca ctg ta - #t aaa gag gcc acg tct 2354 Ala Lys Trp
Asp Thr Ala Asn Asn Pro Leu Ty - #r Lys Glu Ala Thr Ser 765 - # 770
- # 775 - - acc ttc acc aat atc acg tac cgg ggc act ta - #a
tgataagcag tcatcctca g 2407 Thr Phe Thr Asn Ile Thr Tyr Arg Gly Thr
780 - # 785 - - atcattatca gcctgtgcca ggattgcagg agtccctgcc
atcatgttta ca - #gaggacag 2467 - - tatttgtggg gagggatttc ggggctcaga
gtggggtagg ttgggagaat gt - #cagtatgt 2527 - - ggaagtgtgg gtctgtgtgt
gtgtatgtgg gggtctgtgt gtttatgtgt gt - #gtgttgtg 2587 - - tgtgggagtg
tgtaatttaa aattgtgatg tgtcctgata agctgagctc ct - #tagccttt 2647 - -
gtcccagaat gcctcctgca gggattcttc ctgcttagct tgagggtgac ta -
#tggagctg 2707 - - agcaggtgtt cttcattacc tcagtgagaa gccagctttc
ctcatcaggc ca - #ttgtccct 2767 - - gaagagaagg gcagggctga ggcctctcat
tccagaggaa gggacaccaa gc - #cttggctc 2827 - - taccctgagt tcataaattt
atggttctca ggcctgactc tcagcagcta tg - #gtaggaac 2887 - - tgctggcttg
gcagcccggg tcatctgtac ctctgcctcc tttcccctcc ct - #caggccga 2947 - -
aggaggagtc agggagagct gaactattag agctgcctgt gccttttgcc at -
#cccctcaa 3007 - - cccagctatg gttctctcgc aagggaagtc cttgcaagct
aattctttga cc - #tgttggga 3067 - - gtgaggatgt ctgggccact caggggtcat
tcatggcctg ggggatgtac ca - #gcatctcc 3127 - - cagttcataa tcacaaccct
tcagatttgc cttattggca gcg - # 317 - #0 - - - - <210> SEQ ID
NO 2 <211> LENGTH: 23 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: PCR Primer
- - <400> SEQUENCE: 2 - - tttaccactg atgccaagac tca - # - #
23 - - - - <210> SEQ ID NO 3 <211> LENGTH: 21
<212> TYPE: DNA <213> ORGANISM: Artificial Sequence
<220> FEATURE: <223> OTHER INFORMATION: PCR Primer - -
<400> SEQUENCE: 3 - - ccgtcattag gctggacaat g - # - # - #21 -
- - - <210> SEQ ID NO 4 <211> LENGTH: 25 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: PCR Probe - - <400>
SEQUENCE: 4 - - atagcattgg acggaaggct ggcag - # - # 25 - - - -
<210> SEQ ID NO 5 <211> LENGTH: 19 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: PCR Primer - - <400> SEQUENCE:
5 - - gaaggtgaag gtcggagtc - # - # - # 19 - - - - <210> SEQ
ID NO 6 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: PCR Primer - - <400> SEQUENCE: 6 - -
gaagatggtg atgggatttc - # - # - # 20 - - - - <210> SEQ ID NO
7 <211> LENGTH: 20 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: PCR Probe - - <400> SEQUENCE: 7 - -
caagcttccc gttctcagcc - # - # - # 20 - - - - <210> SEQ ID NO
8 <211> LENGTH: 18 <212> TYPE: DNA <213>
ORGANISM: Artificial Sequence <220> FEATURE: <223>
OTHER INFORMATION: Antisense Oligonucleotide - - <400>
SEQUENCE: 8 - - gcatctcgtc cgcctccc - # - # - # 18 - - - -
<210> SEQ ID NO 9 <211> LENGTH: 18 <212> TYPE:
DNA <213> ORGANISM: Artificial Sequence <220> FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide - -
<400> SEQUENCE: 9 - - gccagcacag tcacccag - # - # - # 18 - -
- - <210> SEQ ID NO 10 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 10 - - actcactggg aactcgat - # - # - # 18 -
- - - <210> SEQ ID NO 11 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 11 - - tggatggaga aattcttc - # - # - # 18 -
- - - <210> SEQ ID NO 12 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 12 - - gttctggatg ctccacag - # - # - # 18 -
- - - <210> SEQ ID NO 13 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 13 - - gccccgaagc caatccgc - # - # - # 18 -
- - - <210> SEQ ID NO 14 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 14 - - aagcgggtca cctggtca - # - # - # 18 -
- - - <210> SEQ ID NO 15 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 15 - - cattcctcca gccaatct - # - # - # 18 -
- - - <210> SEQ ID NO 16 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 16 - - cttccgtcca atgctata - # - # - # 18 -
- - - <210> SEQ ID NO 17 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 17 - - tgtcactacc aacatgac - # - # - # 18 -
- - - <210> SEQ ID NO 18 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 18 - - agcttctcag tcatcagc - # - # - # 18 -
- - - <210> SEQ ID NO 19 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 19 - - agagattgac tacatttt - # - # - # 18 -
- - - <210> SEQ ID NO 20 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 20 - - ctttagaacg gattttcc - # - # - # 18 -
- - - <210> SEQ ID NO 21 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 21 - - agacaactct tcagggag - # - # - # 18 -
- - - <210> SEQ ID NO 22 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 22 - - tcaccgtgtc tccaatct - # - # - # 18 -
- - - <210> SEQ ID NO 23 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 23 - - ccacgggctt tatggtaa - # - # - # 18 -
- - - <210> SEQ ID NO 24 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 24 - - tcagcttggg cctggcag - # - # - # 18 -
- - - <210> SEQ ID NO 25 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 25 - - agtcctcctc tgagcact - # - # - # 18 -
- - - <210> SEQ ID NO 26 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 26 - - cacattgacc acagaggc - # - # - # 18 -
- - - <210> SEQ ID NO 27 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 27 - - agaagtcgtc acactcgc - # - # - # 18 -
- - - <210> SEQ ID NO 28 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 28 - - gcccattgct ggacatgc - # - # - # 18 -
- - - <210> SEQ ID NO 29 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE:
<223> OTHER INFORMATION: Antisense Oligonucleotide - -
<400> SEQUENCE: 29 - - aggcatctgg gcaggtgg - # - # - # 18 - -
- - <210> SEQ ID NO 30 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 30 - - tcaaacttct tacactcc - # - # - # 18 -
- - - <210> SEQ ID NO 31 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 31 - - gcatccttgc cagtgtcc - # - # - # 18 -
- - - <210> SEQ ID NO 32 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 32 - - acgacacagt catcctca - # - # - # 18 -
- - - <210> SEQ ID NO 33 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 33 - - accacataca ggatggac - # - # - # 18 -
- - - <210> SEQ ID NO 34 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 34 - - gtgatgagga gtttccag - # - # - # 18 -
- - - <210> SEQ ID NO 35 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 35 - - tgtcccattt tgctctgg - # - # - # 18 -
- - - <210> SEQ ID NO 36 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 36 - - aaggtagacg tggcctct - # - # - # 18 -
- - - <210> SEQ ID NO 37 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 37 - - atgactgctt atcattaa - # - # - # 18 -
- - - <210> SEQ ID NO 38 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 38 - - gactcctgca atcctggc - # - # - # 18 -
- - - <210> SEQ ID NO 39 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 39 - - acatactgac attctccc - # - # - # 18 -
- - - <210> SEQ ID NO 40 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 40 - - acacacacat aaacacac - # - # - # 18 -
- - - <210> SEQ ID NO 41 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 41 - - tcaccctcaa gctaagca - # - # - # 18 -
- - - <210> SEQ ID NO 42 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 42 - - tctcactgag gtaatgaa - # - # - # 18 -
- - - <210> SEQ ID NO 43 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 43 - - agctgctgag agtcaggc - # - # - # 18 -
- - - <210> SEQ ID NO 44 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 44 - - tccttcggcc tgagggag - # - # - # 18 -
- - - <210> SEQ ID NO 45 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 45 - - aaaggcacag gcagctct - # - # - # 18 -
- - - <210> SEQ ID NO 46 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 46 - - agatgctggt acatcccc - # - # - # 18 -
- - - <210> SEQ ID NO 47 <211> LENGTH: 18 <212>
TYPE: DNA <213> ORGANISM: Artificial Sequence <220>
FEATURE: <223> OTHER INFORMATION: Antisense Oligonucleotide -
- <400> SEQUENCE: 47 - - caataaggca aatctgaa - # - # - # 18
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